Antisense modulation of PECAM-1

ABSTRACT

Compositions and methods for the treatment and diagnosis of diseases or disorders amenable to treatment through modulation of expression of a nucleic acid encoding a platelet endothelial cell adhesion molecule-1 (PECAM-1; also known as CD31 antigen or endoCAM) protein are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for detectingand modulating levels of platelet endothelial cell adhesion molecule-1(PECAM-1) proteins, including human PECAM-1 (also known as CD31 antigenor endoCAM). In particular, the invention relates to antisense compoundsspecifically hybridizable with nucleic acids encoding PECAM-1 proteins.It has been found that such antisense compounds can modulate theexpression of PECAM-1 proteins. PECAM-1 proteins are glycoproteins whichare expressed on the surfaces of a variety of cell types (for reviews ofPECAM-1 proteins, see Newman, J. Clin. Invest., 1997, 99, 3 and DeLisseret al., Immunol. Today, 1994, 15, 490). Because PECAM-1 mediatescell-cell interactions, modulation of the expression of PECAM-1 allowsfor the control of such cell-cell interactions and resulting effectssuch as, for example, inflammation. The invention is thus directed todiagnostic methods for detecting, and prophylactic and therapeuticmethods for preventing or inhibiting, respectively, PECAM-1-mediatedprocesses. Furthermore, this invention is directed to treatment ofconditions associated with abnormal expression of PECAM-1 proteins. Thisinvention also relates to therapies, diagnostics, and research reagentsfor disease states or disorders which respond to modulation of theexpression of PECAM-1 proteins. Inhibition of the hyperproliferation ofcells, and corresponding prophylactic, palliative and therapeuticeffects result from treatment with the antisense compounds of theinvention.

BACKGROUND OF THE INVENTION

Cell-cell interactions are a feature of a variety of biologicalprocesses. In the activation of the immune response, for example, one ofthe earliest detectable events in a normal inflammatory response isadhesion of leukocytes to the vascular endothelium, followed bymigration of leukocytes out of the vasculature to the site of infectionor injury. The adhesion of leukocytes to vascular endothelium is anobligate step in their migration out of the vasculature (Harlan, Blood,1985, 65, 515). As is well known in the art, cell-cell interactions arealso critical for propagation of both B-lymphocytes and T-lymphocytesresulting in enhanced humoral and cellular immune responses,respectively.

In several instances, the adhesion of one cell type to another ismediated by interactions between specific proteins, termed "adhesionmolecules," located on the plasma membrane of cells. The interactionbetween adhesion molecules is similar to classical receptor ligandinteractions with the exception that the ligand is fixed to the surfaceof a cell instead of being soluble. One group of related (by peptidesequence), biologically significant molecules mediating cell-cellinteractions are known in the art as CAMs (cellular adhesion molecules).CAMs include, for example, several intercellular adhesion molecules(i.e., ICAM-1, ICAM-2 and ICAM-3), endothelial leukocyte adhesionmolecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1). Thenucleotide and peptide sequences of PECAM-1 (platelet endothelialcellular adhesion molecule 1) indicate that it is also a member of theCAM family of genes. The CAM family is a part of the immunoglobulinsuperfamily of genes (Newman et al., Science, 1990, 247, 1219).

PECAM-1 is a member of the CAM family of proteins and is expressed onthe surfaces of several cell types. Cells known to express PECAM-1include, for example, endothelial cells, including human umbilical veinendothelial cells (HUVEC), bovine aortic endothelial cells (BAEC),glomerular endothelial (GEN) cells and cultured human pulmonarymicrovascular endothelial cells (HPMEC) ; platelets, monocytes,granulocytes, macrophages, some lymphocytes, some hematopoietic lineagecells and tumor cell lines from different mammalian species (seeDeLisser et al., Immunol. Today, 1994, 15, 490.

A variety of evidence indicates that PECAM-1 is involved in cell-cellrecognition events (DeLisser et al., Immunol. Today, 1994, 15, 490). Forexample, PECAM-1 mediates aggregation of L cells transfected withPECAM-1 cDNA (DeLisser et al., J. Biol. Chem., 1993, 268, 16307;) and,in certain T cell subsets, amplifies beta-1 integrin-mediated adhesion(Tanaka et al., J. Exp. Med., 1992, 176, 245). In vitro studies haveimplicated PECAM-1 in the initiation of endothelial cell contact(Albelda et al., J. Cell Biol., 1990, 110, 1227) and capillary tubeformation (Merwin et al., unpublished results cited in Vaporciyan etal., Science, 1993, 262, 1580). In both in vitro and in vivo studies,PECAM-1 has been shown to be involved in the transmigration of whiteblood cells (e.g., neutrophils, monocytes, and other nucleated bloodcells) through endothelial cell monolayers (Muller et al., J. Exp. Med.,1993, 178, 449). In addition to directly participating in cell-cellinteractions, PECAM-1 apparently also regulates the activity and/orexpression of other molecules involved in cellular interactions (Litwinet al., J. Cell Biol., 1997, 139, 219).

Due to PECAM-1's involvement in cellular processes precedinginflammation and tumorigenesis, it is believed that inhibitors ofPECAM-1 expression may provide a novel therapeutic class ofimmunosuppressive and/or anti-inflammatory and anticancer agents withactivity towards (1) autoimmune disorders such as multiple sclerosis,particularly autoimmune disorders of the thyroid such as Graves'disease, and undesired immune responses, such as, for example, thosethat occur in graft versus host disease (GVHD); (2) a variety ofinflammatory diseases or disorders with an inflammatory component suchas various forms of arthritis; allograft rejections; inflammatorydiseases of the bowel, including Crohn's disease; various dermatologicalconditions such as psoriasis; corneal inflammation and the like, and (3)a variety of hyperproliferative diseases or disorders including, but notlimited to, cancers, tumors, and the growth and spreading (metastasis)thereof.

To date, there are no known therapeutic agents which effectively preventthe expression of PECAM-1. Current agents which affect intercellularadhesion molecules include synthetic peptides, monoclonal antibodies,and soluble forms of the adhesion molecules. Synthetic peptides whichblock cellular interactions with PECAM-1 by mimicking a ligand ofPECAM-1 have not been identified, although a peptidic mimic of PECAM-1has been described (Liao et al., J. Exp. Med., 1997, 185, 1349).Monoclonal antibodies to PECAM-1 have been developed (Lastres et al., J.Immunol., 1994, 153, 4206) and may prove to be useful for the treatmentof acute inflammatory responses or other conditions resulting fromexpression of PECAM-1 (Bogen et al., J. Exp. Med., 1994, 179, 1059;Murohara et al., J. Immunol., 1996, 156, 3550). However, with chronictreatment, the host animal can develop antibodies against the monoclonalantibodies themselves, thereby limiting their usefulness. In addition,monoclonal antibodies are large proteins which may have difficulty ingaining access to inflammatory sites. Soluble forms of the cell adhesionmolecules suffer from many of the same limitations as monoclonalantibodies in addition to the expense of their production and their lowbinding affinity. All three of the above types of agents arepolypeptides and are subject to proteolytic degradation in vivo, afeature which may limit the biological half-life and pharmacologicaleffectiveness. Moreover, due to the high degree of homology betweenPECAM-1 and other members of the CAM family at the polypeptide level,agents which effect PECAM-1 activity might also effect other CAMproteins. Thus, there is a long felt need for molecules whicheffectively and specifically modulate PECAM-1 molecules.

Antisense oligonucleotides avoid many of the pitfalls of current agentsused to block the effects of PECAM-1. For example, antisenseoligonucleotides are smaller than monoclonal antibodies and mostsynthetic peptides and are thus expected to have better access to sitesof inflammation. A variety of chemical modifications are known whichserve to enhance the affinity of an antisense oligonucleotide for itstarget nucleic acid. Other chemical modifications have been describedthat render synthetic oligonucleotides more resistant to in vitro and invivo degradation. Moreover, because of the degeneracy inherent in thegenetic code, the homology between the nucleotide sequence of a nucleicacid which encodes a PECAM-1 protein and those nucleotide sequencesencoding other CAM proteins will be less than the homology between thecorresponding polypeptide sequences; as a result, antisenseoligonucleotides have the potential to achieve a higher degree ofspecificity than many monoclonal antibodies. In short, the compounds ofthe invention are particularly effective for specifically modulatingPECAM-1 molecules.

RELATED ART

U.S. Pat. No. 5,264,554 to Newman describes PECAM-1 protein and variantsthereof. See also PCT application WO 91/10683 published Jul. 25, 1991.

U.S. Pat. No. 5,668,012 to Newman et al. describes PECAM-1 promotersequences and sequences encoding PECAM-1 isoforms. See also PCTapplication WO 96/01271 published Jan. 18, 1996.

PCT application WO 97/10839, published Mar. 27, 1997, describessynthetic peptides stated to inhibit binding of PECAM-1 monomers to eachother.

Liao et al. (J. Exp. Med., 1997, 185, 1349) describe fusion proteinscomprising the soluble domain 1 of PECAM-1 and indicate that thesefusion proteins are sufficient to modulate diapedesis in vivo and invitro.

Berman et al. (J. Immunol., 1996, 1516, 1515) describe antibodies toPECAM-1 that are stated to modulate cellular transmigration events invitro.

Murohara et al. (J. Immunol., 1996, 156, 3550) describe a monoclonalantibody to human and feline PECAM-1 that is stated to inhibit leukocytemigration in glycogen-induced peritonitis in vivo in cats.

Lastres et al. (J. Immunol., 1994, 153, 4206) describe antibodies and aphosphorothioate antisense oligonucleotide targeted to the start (ATG)codon of PECAM-1 that inhibits cellular aggregation in an in vitroassay. PECAM-1 mRNA or protein levels following treatment were notdetermined.

Bogen et al. (J. Exp. Med., 1994, 179, 1059) describe a monoclonalantibody to murine PECAM-1 and indicate that it modulates acuteinflammation in thioglycollate-induced peritonitis in mice.

Vaporciyan et al. (Science, 1993, 262, 1580) describe a monoclonalantibody to human PECAM-1 that cross-reacts with rat PECAM-1 and whichis stated to modulate neutrophil transmigration in rats.

Tang et al. (J. Biol. Chem., 1993, 268, 22883) describe antibodies toPECAM-1 stated to have the ability to decrease the in vitro adhesion oftumor cells to microvascular endothelial cells.

SUMMARY OF THE INVENTION

In accordance with the present invention, antisense compounds areprovided which specifically hybridize with a nucleic acid encoding aPECAM-1 protein. Certain antisense compounds of the invention aredesigned to bind either directly to mRNA transcribed from, or to aselected DNA portion of, a gene that encodes a PECAM-1 protein, therebymodulating the expression thereof. In particular embodiments of theinvention, the PECAM-1 protein, and the gene encoding it, are those of amammal including a human. Pharmaceutical compositions comprising theantisense compounds of the invention, and various methods of using theantisense compounds of the invention, are also herein provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show the sequence of a cDNA encoding human PECAM-1 (SEQ IDNO: 1; Genbank Accession No. M28526, locus name "HUMPECAM1") and thelocations and sequences of antisense oligonucleotides described in theExamples. The cDNA sequence is written from 5' to 3' and has verticalmarks indicating every 10th base; the cumulative number of bases in thesequence is given in bold to the right of each line. The start (ATG) andstop (TAG) codons are emboldened and double-underlined. The nucleotidebase sequences of the antisense oligonucleotides are written from 3' to5' to demonstrate their complementary nature, and their ISIS number isgiven in parentheses to the right of each line.

FIG. 2 shows a portion of the sequence of the stop codon region of acDNA encoding human PECAM-1 (bases 2305-2394 of SEQ ID NO: 1; GenBankAccession No. M28526, locus name "HUMPECAM1") and the locations andsequences of various antisense oligonucleotides described in theExamples. The cDNA sequence is written from 3' to 5', base 2350 isindicated by a vertical mark, and the stop codon is emboldened andunderlined. The nucleotide base sequences of the antisenseoligonucleotides are written from 5' to 3'.

FIG. 3 shows a portion of the sequence of the 3' untranslated region ofa cDNA encoding human PECAM-1 (bases 2436-2520 of SEQ ID NO: 1) and thelocations and sequences of various antisense oligonucleotides describedin the Examples. The cDNA sequence is written from 3' to 5' and bases2450 and 2500 are indicated by vertical marks. The nucleotide basesequences of the antisense oligonucleotides are written from 5' to 3'.

DETAILED DESCRIPTION OF THE INVENTION

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides are commonly described as"antisense." Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. It has beendiscovered that genes encoding platelet endothelial cell adhesionmolecule-1 (PECAM-1; also known as CD31 antigen or endoCAM) proteins,including human PECAM-1, are particularly amenable to this approach.More specifically, the present invention is directed to antisensecompounds, including oligonucleotides, that modulate the expression ofPECAM-1.

Methods of modulating the expression of PECAM-1 proteins with antisensecompounds are provided herein and are believed to be useful boththerapeutically and diagnostically as a consequence of the associationbetween PECAM-1 expression and certain hyperproliferative andinflammatory disorders. These methods are also useful as tools, forexample, in the detection and determination of the role of PECAM-1 invarious cell functions and physiological processes and conditions, andfor the diagnosis of conditions associated with such expression andactivation.

As a consequence of the association between PECAM-1 and normal andabnormal cell-cell interactions, inhibition of the expression of PECAM-1proteins is expected to lead to, for example, the inhibition of avariety of inflammatory events and tumorigenic and/or metastatic eventsand, accordingly, results in modulation of the undesirable consequencesof such events. Such modulation is desirable for treating (i.e.,providing prophylactic, palliative and/or therapeutic effects) variousinflammatory and hyperproliferative disorders or diseases. Suchinhibition of PECAM-1, and other CAMs, is further desirable forpreventing or modulating the development of such diseases or disordersin an animal suspected of being, or known to be, prone to such diseasesor disorders.

The present invention also comprises methods of inhibiting a variety ofPECAM-1-mediated inflammatory and tumorigenic and/or metastatic eventsusing the antisense compounds of the invention. Methods of treatingconditions in which abnormal or excessive PECAM-1 expression and/orPECAM-1-mediated inflammation occurs are also provided. These methodsemploy the antisense compounds of the invention and are believed to beuseful both therapeutically and as clinical research and diagnostictools. The oligonucleotides of the present invention may also be usedfor research purposes. Thus, the specific hybridization exhibited by theoligonucleotides of the present invention may be used for assays,purifications, cellular product preparations and in other methodologieswhich may be appreciated by persons of ordinary skill in the art.

The present invention employs antisense compounds which modulate thefunction of DNA or messenger RNA (mRNA) encoding a protein (PECAM-1) themodulation of which is desired and ultimately regulate the expression ofthe protein. Hybridization of an antisense oligonucleotide with its mRNAtarget interferes with the normal role of mRNA and causes a modulationof its function in cells. The functions of mRNA to be interfered withinclude all vital functions such as translocation of the RNA to the sitefor protein translation, actual translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and possibly evenindependent catalytic activity which may be engaged in by the RNA. Theoverall effect of such interference with mRNA function is modulation ofthe expression of a protein, wherein "modulation" means either anincrease (stimulation) or a decrease (inhibition) in the expression ofthe protein. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression.

It is preferred to target specific genes for antisense attack."Targeting" an oligonucleotide to the associated nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene associated with hyperproliferative disorders. The targeting processalso includes determination of a site or sites within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult. Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity to give the desired effect. Generally, there are severalregions of a gene that may be targeted for antisense modulation: the 5'"cap," which comprises an N7-methylated guanosine residue joined to themost 5' residue of the mRNA via a triphosphate linkage (Baker, Chapter 3In: Antisense Research and Applications, Crooke et al., eds., CRC Press,Boca Raton, Fla., 1993, pages 37-53); the 5' untranslated region(hereinafter, the "5'-UTR"), the translation initiation codon region(hereinafter, the "AUG" region), the open reading frame (hereinafter,the "ORF") or "coding region," the translation termination codon region(hereinafter, the "stop codon" region or simply "stop" for short) andthe 3' untranslated region (hereinafter, the "3'-UTR"). As is known inthe art, these regions are arranged in a typical messenger RNA moleculein the following order (left to right, 5' to 3'): cap, 5'-UTR, AUG, ORF,stop codon, 3'-UTR. As is known in the art, although some eukaryotictranscripts are directly translated, many ORFs contain one or moresequences, known as "introns," which are excised from a transcriptbefore it is translated; the expressed (unexcised) portions of the ORFare referred to as "exons" (Alberts et al., Molecular Biology of theCell, 1983, Garland Publishing Inc., New York, pp. 411-415).Furthermore, because many eukaryotic ORFs are a thousand nucleotides ormore in length, it is often convenient to subdivide the ORF into, e.g.,the 5' ORF region, the central ORF region, and the 3' ORF region. Insome instances, an ORF contains one or more sites that may be targeteddue to some functional significance in vivo. Examples of the lattertypes of sites include intragenic stem-loop structures (see, e.g., U.S.Pat. No. 5,512,438) and, in unprocessed mRNA molecules, intron/exonsplice sites.

Within the context of the present invention, one preferred intragenicsite is the region encompassing the translation initiation codon of theopen reading frame (ORF) of the gene. Because, as is known in the art,the translation initiation codon is typically 5'-AUG (in transcribedmRNA molecules; 5'-ATG in the corresponding DNA molecule), thetranslation initiation codon is also referred to as the "AUG codon," the"start codon" or the "AUG start codon." A minority of genes have atranslation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function invivo. Furthermore, 5'-UUU functions as a translation initiation codon invitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al.,Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, Vol. 2, 1987, Neidhardt et al., eds., American Society forMicrobiology, Washington, D.C., p. 1303). Thus, the terms "translationinitiation codon" and "start codon" can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (prokaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes may have two ormore alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions, in order to generate relatedpolypeptides having different amino terminal sequences. In the contextof the invention, "start codon" and "translation initiation codon" referto the codon or codons that are used in vivo to initiate translation ofan mRNA molecule transcribed from a gene encoding a PECAM-1 protein,regardless of the nucleotide sequence(s) of such codons. It is alsoknown in the art that a translation termination codon (or "stop codon")of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA,respectively). The terms "start codon region" and "translationinitiation region" refer to a portion of such an mRNA or gene thatencompasses about 50 contiguous (adjacent) nucleotides in eitherdirection (i.e., 5' or 3') from a translation initiation codon.Similarly, the terms "stop codon region" and "translation terminationregion" refer to a portion of such an mRNA or gene that encompassesabout 50 contiguous (adjacent) nucleotides in either direction (i.e., 5'or 3') from a translation termination codon.

The remainder of the Detailed Description relates in more detail the (1)Antisense Compounds of the Invention and (2) Bioequivalents and (3)Exemplary Utilities thereof, as well as (4) Pharmaceutical Compositionscomprising the Antisense Compounds of the Invention and (5) Methods ofAdministration thereof.

1. Antisense Compounds of the Invention: The present invention employsantisense compounds that modulate PECAM-1 proteins. The term "antisensecompounds" (a) specifically includes synthetic oligonucleotides, as wellas peptide nucleic acids (PNAs), having a nucleobase sequencespecifically hybridizable with a nucleic acid encoding a PECAM-1 proteinand (b) specifically excludes ribozymes and nucleic acids of biologicalorigin. In the context of this invention, the term "oligonucleotide"refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent intersugar (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. The antisense compounds of theinvention are synthesized in vitro and do not include antisensecompositions of biological origin, or genetic vector constructs designedto direct the in vivo synthesis of antisense molecules.

The antisense compounds in accordance with this invention preferablycomprise from about 8 to about 30 nucleobases, more preferably fromabout 12 to about 28 and most preferably from about 20 to about 26nucleobases. Particularly preferred antisense compounds are antisenseoligonucleotides. A discussion of antisense oligonucleotides and somedesirable modifications can be found in De Mesmaeker et al., Acc. Chem.Res., 1995, 28, 366.

An oligonucleotide is a polymer of a repeating unit generically known asa nucleotide. An unmodified (naturally occurring) nucleotide has threecomponents: (1) a nitrogen-containing heterocyclic base linked by one ofits nitrogen atoms to (2) a 5-pentofuranosyl sugar and (3) a phosphateesterified to one of the 5' or 3' carbon atoms of the sugar. Whenincorporated into an oligonucleotide chain, the phosphate of a firstnucleotide is also esterified to an adjacent sugar of a second, adjacentnucleotide via a 3'-5' phosphate linkage.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2', 3' or 5' hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be further joined to form a circular structure, however,within the context of the invention, open linear structures aregenerally preferred.

Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the intersugar "backbone" of the oligonucleotide.The normal linkage or backbone of RNA and DNA is a 3' to 5'phosphodiester linkage. The backbone of an oligonucleotide (or otherantisense compound) positions a series of bases in a specific order; thewritten representation of this ordered series of bases, usually writtenin 5' to 3' order unless otherwise indicated, is known as a nucleotideor nucleobase sequence.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as"antisense." In the context of the invention, "hybridization" meanshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. "Complementary," as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother.

"Specifically hybridizable" and "complementary" are thus terms which areused to indicate a sufficient degree of complementarity or precisepairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. An oligonucleotide isspecifically hybridizable to its target sequence due to the formation ofbase pairs between specific partner nucleobases in the interior of anucleic acid duplex. Among the naturally occurring nucleobases, guanine(G) binds to cytosine (C), and adenine (A) binds to thymine (T) oruracil (U). In addition to the equivalency of U (RNA) and T (DNA) aspartners for A, other naturally occurring nucleobase equivalents areknown, including 5-methylcytosine and 5-hydroxymethylcytosine (HMC) (Cequivalents), and 5-hydroxymethyluracil (U equivalent). Furthermore,synthetic nucleobases which retain partner specificity are known in theart and include, for example, 7-deaza-Guanine, which retains partnerspecificity for C. Thus, an oligonucleotide's capacity to specificallyhybridize with its target sequence will not be altered by a chemicalmodification to a nucleobase in the nucleotide sequence of theoligonucleotide which does not impact its specificity for a partnernucleobase in the target nucleic acid.

It is understood in the art that the nucleobase sequence of anoligonucleotide or other antisense compound need not be 100%.complementary to its target nucleic acid sequence to be specificallyhybridizable. An antisense compound is specifically hybridizable to itstarget nucleic acid when there is a sufficient degree of complementarityto avoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, or, in the case of in vitro assays, under assayconditions.

Antisense oligonucleotides are commonly used as research reagents,diagnostic aids, and therapeutic agents. For example, antisenseoligonucleotides, which are able to inhibit gene expression withexquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses. The specificity and sensitivity ofoligonucleotides is also harnessed by those of skill in the art fortherapeutic uses. Specific examples of preferred antisense compoundsuseful in this invention include oligonucleotides containing modifiedbackbones or non-natural intersugar linkages. As defined in thisspecification, oligonucleotides having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modifiedoligonucleotides that do not have a phosphorus atom in their intersugarbackbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described in thefollowing subsections. It is not necessary for all positions in a givencompound to be uniformly modified, and in fact more than one of thefollowing modifications may be incorporated in a single antisensecompound or even in a single residue thereof, for example, at a singlenucleoside within an oligonucleotide.

A. Modified Linkages: Preferred modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3'-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3'-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalklyphosphotriesters, and boranophosphates having normal 3'-5'linkages, 2'-5' linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3'-5'to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acidforms are also included.

Representative United States Patents that teach the preparation of theabove phosphorus atom containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein (i.e., oligonucleosides) have backbones that areformed by short chain alkyl or cycloalkyl intersugar linkages, mixedheteroatom and alkyl or cycloalkyl intersugar linkages, or one or moreshort chain heteroatomic or heterocyclic intersugar linkages. Theseinclude those having morpholino linkages (formed in part from the sugarportion of a nucleoside); siloxane backbones; sulfide, sulfoxide andsulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene containing backbones;sulfamate backbones; methyleneimino and methylenehydrazino backbones;sulfonate and sulfonamide backbones; amide backbones; and others havingmixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theintersugar linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂--N(CH₃)--O--CH₂ -- known as a methylene (methylimino) or MMI backbone!,--CH₂ --O--N(CH₃)--CH₂ --, --CH₂ --N(CH₃)-- N(CH₃)--CH₂ -- and--O--N(CH₃)--CH₂ --CH₂ -- wherein the native phosphodiester backbone isrepresented as --O--P--O--CH₂ --! of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

B. Modified Nucleobases: The compounds of the invention may additionallyor alternatively comprise nucleobase (often referred to in the artsimply as "base") modifications or substitutions. As used herein,"unmodified" or "natural" nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5--me--C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Id., pages276-278) and are presently preferred base substitutions, even moreparticularly when combined with 2'-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/762,488, filed onDec. 10, 1996, also herein incorporated by reference.

C. Sugar Modifications: The antisense compounds of the invention mayadditionally or alternatively comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O,S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O (CH₂)_(n) O!_(m) CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂, and O(CH₂)_(n) ON(CH₂)_(n) CH₃)!₂, where n and m are from 1 to about 10. Other preferredoligonucleotides comprise one of the following at the 2' position: C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide, and other substituentshaving similar properties. A preferred modification includes2'-methoxyethoxy 2'--O--CH₂ CH₂ OCH₃, also known as2'--O--(2-methoxyethyl) or 2'-MOE! (Martin et al., Helv. Chim. Acta,1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE, as described in co-owned U.S.patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, thecontents of which are herein incorporated by reference.

Other preferred modifications include 2'-methoxy (2'--O--CH₃),2'-aminopropoxy (2'--OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'--F) Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'position of 5' terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/468,037, filed onJun. 5, 1995, also herein incorporated by reference.

D. Other Modifications: Additional modifications may also be made atother positions on the oligonucleotide, particularly the 3' position ofthe sugar on the 3' terminal nucleotide and the 5' position of 5'terminal nucleotide. For example, one additional modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327;Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned, and each of which is herein incorporated byreference.

E. Chimeric Oligonucleotides: The present invention also includesantisense compounds which are chimeric compounds. "Chimeric" antisensecompounds or "chimeras," in the context of this invention, are antisensecompounds, particularly oligonucleotides, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart. RNase H-mediated target cleavage is distinct from the use ofribozymes to cleave nucleic acids, and ribozymes are not comprehended bythe present invention.

By way of example, such "chimeras" may be "gapmers," i.e.,oligonucleotides in which a central portion (the "gap") of theoligonucleotide serves as a substrate for, e.g., RNase H, and the 5' and3' portions (the "wings") are modified in such a fashion so as to havegreater affinity for, or stability when duplexed with, the target RNAmolecule but are unable to support nuclease activity (e.g., 2'-fluoro-or 2'-methoxyethoxy- substituted). Other chimeras include "hemimers,"that is, oligonucleotides in which the 5' portion of the oligonucleotideserves as a substrate for, e.g., RNase H, whereas the 3' portion ismodified in such a fashion so as to have greater affinity for, orstability when duplexed with, the target RNA molecule but is unable tosupport nuclease activity (e.g., 2'-fluoro- or 2'-methoxyethoxy-substituted), or vice-versa.

A number of chemical modifications to oligonucleotides that confergreater oligonucleotide:RNA duplex stability have been described byFreier et al. (Nucl. Acids Res., 1997, 25, 4429). Such modifications arepreferred for the RNase H-refractory portions of chimericoligonucleotides and may generally be used to enhance the affinity of anantisense compound for a target RNA.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned, and each of which is hereinincorporated by reference, and commonly owned and allowed U.S. patentapplication Ser. No. 08/465,880, filed on Jun. 6, 1995, also hereinincorporated by reference.

F. Synthesis: The oligonucleotides used in accordance with thisinvention may be conveniently and routinely made through the well-knowntechnique of solid phase synthesis. Equipment for such synthesis is soldby several vendors including, for example, Applied Biosystems (FosterCity, Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

1. Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents or pendingpatent applications, each of which is commonly assigned with thisapplication: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2'-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.Nos. 5,223,168, issued Jun. 29, 1993, and 5,608,046, both drawn toconjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240,and 5,610,289, drawn to backbone modified oligonucleotide analogs; andU.S. patent application Ser. No. 08/383,666, filed Feb. 3, 1995, andU.S. Pat. No. 5,459,255, drawn to, inter alia, methods of synthesizing2'-fluoro-oligonucleotides.

2. Bioequivalents: The compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to "prodrugs" and "pharmaceutically acceptablesalts" of the antisense compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

A. oligonucleotide Prodrugs: The antisense compounds of the inventionmay additionally or alternatively be prepared to be delivered in a"prodrug" form. The term "prodrug" indicates a therapeutic agent that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. In particular, prodrugversions of the antisense compounds of the invention are prepared asSATE (S-acetyl-2-thioethyl) phosphate! derivatives according to themethods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9,1993.

B. Pharmaceutically Acceptable Salts: The term "pharmaceuticallyacceptable salts" refers to physiologically and pharmaceuticallyacceptable salts of the antisense compounds of the invention: i.e.,salts that retain the desired biological activity of the parent compoundand do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines are chloroprocaine,choline, N,N'-dibenzylethylenediamine, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., "Pharmaceutical Salts," J. of PharmaSci., 1977, 66:1). The base addition salts of said acidic compounds areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a "pharmaceutical addition salt"includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid, embonicacid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, nicotinic acid,isonicotinic acid or 2-acetoxybenzoic acid; and with amino acids, suchas the 20 alpha-amino acids involved in the synthesis of proteins innature, for example glutamic acid or aspartic acid, and also withnaphthalene-1,5-disulfonic acid, phenylacetic acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, methanesulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

3. Exemplary Utilities of the Invention: The oligonucleotides of thepresent invention specifically hybridize to nucleic acids (e.g., mRNAs)encoding a PECAM-1 protein. The antisense compounds of the presentinvention can be utilized as therapeutic compounds, as diagnostic toolsor research reagents that can be incorporated into kits as well as othermethodologies as will be apparent to persons of ordinary skill in theart.

A. Assays and Diagnostic Applications: The oligonucleotides of thepresent invention can be used to detect the presence of PECAM-1protein-specific nucleic acids in a cell or tissue sample. For example,radiolabeled oligonucleotides can be prepared by ³² p labeling at the 5'end with polynucleotide kinase. (Sambrook et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 10.59.) Radiolabeled oligonucleotides are then contacted with cellor tissue samples suspected of containing PECAM-1 protein message RNAs(and thus PECAM-1 proteins), and the samples are washed to removeunbound oligonucleotide. Radioactivity remaining in the sample indicatesthe presence of bound oligonucleotide, which in turn indicates thepresence of nucleic acids complementary to the oligonucleotide, and canbe quantitated using a scintillation counter or other routine means.Expression of nucleic acids encoding these proteins is thus detected.

Radiolabeled oligonucleotides of the present invention can also be usedto perform autoradiography of tissues to determine the localization,distribution and quantitation of PECAM-1 proteins for research,diagnostic or therapeutic purposes. In such studies, tissue sections aretreated with radiolabeled oligonucleotide and washed as described above,then exposed to photographic emulsion according to routineautoradiography procedures. The emulsion, when developed, yields animage of silver grains over the regions expressing a PECAM-1 proteingene. Quantitation of the silver grains permits detection of theexpression of mRNA molecules encoding these proteins and permitstargeting of oligonucleotides to these areas.

Analogous assays for fluorescent detection of expression of PECAM-1protein nucleic acids can be developed using oligonucleotides of thepresent invention which are conjugated with fluorescein or otherfluorescent tags instead of radiolabeling. Such conjugations areroutinely accomplished during solid phase synthesis usingfluorescently-labeled amidites or controlled pore glass (CPG) columns.Fluorescein-labeled amidites and CPG are available from, e.g., GlenResearch, Sterling Va. Other means of labeling oligonucleotides areknown in the art (see, e.g., Ruth, Chapter 6 In: Methods in MolecularBiology, Vol. 26: Protocols for Oligonucleotide Conjugates, Agrawal,ed., Humana Press Inc., Totowa, N.J., 1994, pages 167-185).

Kits for detecting the presence or absence of expression of a PECAM-1protein may also be prepared. Such kits include an oligonucleotidetargeted to an appropriate gene, i.e., a gene encoding a PECAM-1protein. Appropriate kit and assay formats, such as, e.g., "sandwich"assays, are known in the art and can easily be adapted for use with theantisense compounds of the invention. Hybridization of the antisensecompounds of the invention with a nucleic acid encoding a PECAM-1protein can be detected by means known in the art. Such means mayinclude conjugation of an enzyme to the oligonucleotide, radiolabellingof the oligonucleotide or any other suitable detection systems.

B. Biologically Active Oligonucleotides: The invention is also drawn tothe administration of oligonucleotides having biological activity tocultured cells, isolated tissues and organs and animals. By "havingbiological activity," it is meant that the oligonucleotide functions tomodulate the expression of one or more genes in cultured cells, isolatedtissues or organs and/or animals. Such modulation can be achieved by anantisense oligonucleotide by a variety of mechanisms known in the art,including but not limited to transcriptional arrest; effects on RNAprocessing (capping, polyadenylation and splicing) and transportation;enhancement of cellular degradation of the target nucleic acid; andtranslational arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996,6:855).

In an animal other than a human, the compositions and methods of theinvention can be used to study the function of one or more genes in theanimal. For example, antisense oligonucleotides have been systemicallyadministered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-α, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al., Nature, 1993, 363:260; Dean et al., Proc. Natl.Acad. Sci. U.S.A., 1994, 91:11762; and Wahlestedt et al., Science, 1993,259:528, respectively). In instances where complex families of relatedproteins are being investigated, "antisense knockouts" (i.e., inhibitionof a gene by systemic administration of antisense oligonucleotides) mayrepresent the most accurate means for examining a specific member of thefamily (see, generally, Albert et al., Trends Pharmacol. Sci., 1994,15:250).

The compositions and methods of the invention also have therapeutic usesin an animal, including a human, having (i.e., suffering from), or knownto be or suspected of being prone to having, a disease or disorder thatis treatable in whole or in part with one or more nucleic acids. Theterm "therapeutic uses" is intended to encompass prophylactic,palliative and curative uses wherein the antisense compounds of theinvention are contacted with animal cells either in vivo or ex vivo.When contacted with animal cells ex vivo, a therapeutic use includesincorporating such cells into an animal after treatment with one or moreof the antisense compounds of the invention.

For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a PECAM-1 protein is, for example, treated byadministering oligonucleotides in accordance with this invention. Theantisense compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an oligonucleotide to asuitable pharmaceutically acceptable carrier such as, e.g., a diluent.Workers in the field have identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans. The following U.S. patentsdemonstrate palliative, therapeutic and other methods utilizingantisense oligonucleotides. U.S. Pat. No. 5,135,917 provides antisenseoligonucleotides that inhibit human interleukin-1 receptor expression.U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotidescomplementary to the c-myb oncogene and antisense oligonucleotidetherapies for certain cancerous conditions. U.S. Pat. No. 5,087,617provides methods for treating cancer patients with antisenseoligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428provides antisense oligonucleotides having antiviral activity againstinfluenzavirus. U.S. Pat. No. 4,806,463 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementarybase sequence to a portion of an oncogene. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotideanalogs used to prevent replication of foreign nucleic acids in cells.U.S. Pat. No. 4,689,320 is directed to antisense oligonucleotides asantiviral agents specific to cytomegalovirus (CMV). U.S. Pat. No.5,098,890 provides oligonucleotides complementary to at least a portionof the mRNA transcript of the human c-myb gene. U.S. Pat. No. 5,242,906provides antisense oligonucleotides useful in the treatment of latentEpstein-Barr virus (EBV) infections.

As used herein, the term "disease or disorder" (1) includes any abnormalcondition of an organism or part, especially as a consequence ofinfection, inherent weakness, environmental stress, that impairs normalphysiological functioning; (2) excludes pregnancy per se but notautoimmune and other diseases associated with pregnancy; and (3)includes cancers and tumors. The term "known to be or suspected of beingprone to having a disease or disorder" indicates that the subject animalhas been determined to be, or is suspected of being, at increased risk,relative to the general population of such animals, of developing aparticular disease or disorder as herein defined. For example, a subjectanimal "known to be or suspected of being prone to having a disease ordisorder" could have a personal and/or family medical history thatincludes frequent occurrences of a particular disease or disorder. Asanother example, a subject animal "known to be or suspected of beingprone to having a disease or disorder" could have had such asusceptibility determined by genetic screening according to techniquesknown in the art (see, e.g., U.S. Congress, Office of TechnologyAssessment, Chapter 5 In: Genetic Monitoring and Screening in theWorkplace, OTA-BA-455, U.S. Government Printing Office, Washington,D.C., 1990, pages 75-99). The term "a disease or disorder that istreatable in whole or in part with one or more antisense compounds"refers to a disease or disorder, as herein defined, (1) the management,modulation or treatment thereof, and/or (2) therapeutic, curative,palliative and/or prophylactic relief therefrom, can be provided via theadministration of compositions comprising one or more antisensecompounds of the invention.

4. Pharmaceutical Compositions Comprising Compounds of the Invention:The present invention provides for therapeutic and pharmaceuticalcompositions comprising one or more PECAM-1-modulating antisensecompounds. Compositions for the administration of the antisensecompounds of the invention may include sterile aqueous solutions whichmay also contain buffers, diluents and other suitable additives.

A. Compositions for Alimentary Delivery: In a preferred embodiment ofthe invention, one or more PECAM-1-modulating antisense compounds areadministered via alimentary delivery, preferably by oral administration.Pharmaceutical compositions for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets, troches, tablets or SECs (soft elastic capsules or"caplets"). Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, carrier substances or binders may be added to suchcompositions. Such pharmaceutical compositions have the effect ofdelivering the antisense compound(s) to the alimentary canal forexposure to the mucosa thereof. Accordingly, the pharmaceuticalcomposition can comprise material effective in protecting theoligonucleotide from pH extremes of the stomach, or in releasing theoligonucleotide over time, to optimize the delivery thereof to aparticular mucosal site. Enteric coatings for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol and sorbitan monoleate. Various methods forproducing pharmaceutical compositions for alimentary delivery are wellknown in the art. See, generally, Nairn, Chapter 83; Block, Chapter 87;Rudnic et al., Chapter 89; Porter, Chapter 90; and Longer et al.,Chapter 91 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990.

The antisense compounds of the invention can be incorporated in a knownmanner into customary pharmaceutical compositions, such as tablets,coated tablets, pills, granules, aerosols, syrups, emulsions,suspensions and solutions, using inert, non-toxic, pharmaceuticallyacceptable carriers (excipients). The therapeutically active compoundshould in each case be present here in a concentration of about 0.5% toabout 95% by weight of the total mixture, i.e., in amounts which aresufficient to achieve the stated dosage range. The pharmaceuticalcompositions are prepared, for example, by diluting the active compoundswith pharmaceutically acceptable carriers, if appropriate usingemulsifying agents and/or dispersing agents, and, for example, in thecase where water is used as the diluent, organic solvents can be used asauxiliary solvents if appropriate. Pharmaceutical compositions may beformulated in a conventional manner using additional pharmaceuticallyacceptable carriers as appropriate. Thus, the compositions may beprepared by conventional means with additional excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrates (e.g., starchor sodium starch glycolate); or wetting agents (e.g., sodium laurylsulfate). Tablets are coated by methods well known in the art and mayalso contain flavoring, coloring and/or sweetening agents.

Compositions comprising one or more PECAM-1-modulating antisensecompounds can be administered via the rectal mode. In particular,therapeutic or pharmaceutical compositions for rectal administrationinclude foams, solutions (enemas) and suppositories. Rectalsuppositories for adults are usually tapered at one or both ends andtypically weigh about 2 g each, with infant rectal suppositoriestypically weighing about one-half as much when the usual base, cocoabutter, is used (Block, Chapter 87 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990).

The pharmaceutical compositions, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredient(s) with thepharmaceutically acceptable carrier(s). In general the pharmaceuticalcompositions are prepared by uniformly and intimately bringing intoassociation the active ingredient(s) with liquid excipients or finelydivided solid excipients or both, and then, if necessary, shaping theproduct.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing predetermined amounts of the activeingredients; as powders or granules; as solutions or suspensions in anaqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions orwater-in-oil liquid emulsions. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein. Sustained releaseoral delivery systems and/or enteric coatings for orally administereddosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552;4,309,406; and 4,309,404.

B. Additives: Pharmaceutical and therapeutic compositions comprising oneor more of the antisense compounds of the invention may further includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives. Pharmaceutically acceptable organic orinorganic carrier substances suitable for non-parenteral administrationwhich do not deleteriously react with the antisense compounds can beused. The pharmaceutical compositions can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings flavorings and/or aromatic substances andthe like which do not deleteriously react with the oligonucleotide(s) ofthe pharmaceutical composition. Pharmaceutical compositions in the formof aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. Optionally, suchcompositions may also contain one or more stabilizers, penetrationenhancers, carrier compounds or pharmaceutically acceptable carriers.

(1) Penetration Enhancers: Pharmaceutical compositions comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8:91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1).

Fatty Acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, acylcarnitines, acylcholines,1-dodecylazacycloheptan-2-one, mono- and di-glycerides andphysiologically acceptable salts thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7:1; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44:651).

Bile Salts: The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, "bile salt"includes any of the naturally occurring components of bile and any oftheir synthetic derivatives.

Chelating Agents: Chelating agents have the added advantage of alsoserving as DNase inhibitors and include, but are not limited to, citricacid, disodium ethylenediaminetetraacetate (EDTA), salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Crit. Rev. Therap. Drug CarrierSystems, 1991, p. 92; Muranishi, Crit. Rev. Therap. Drug CarrierSystems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14, 43).

Surfactants: Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol., 1988, 40:252).

Non-Surfactants: Non-surfactants include, for example, unsaturatedcyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39:621).

(2) Carrier Compounds: As used herein, "carrier compound" refers to anucleic acid, or analog thereof, which is inert (i.e., does not possessbiological activity per se) but is recognized as a nucleic acid by invivo processes that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioatedoligonucleotide in hepatic tissue is reduced when it is coadministeredwith polyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5:115; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6:177).

(3) Pharmaceutically Acceptable Carriers: In contrast to a carriercompound, a "pharmaceutically acceptable carrier" (excipient) is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering one or more nucleic acidsto an animal. The pharmaceutically acceptable carrier may be liquid orsolid and is selected with the planned manner of administration in mindso as to provide for the desired bulk, consistency, etc., when combinedwith a nucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.). Suitable pharmaceutically acceptable carriers include,but are not limited to, water, salt solutions, alcohol, polyethyleneglycols, gelatin, lactose, amylose, magnesium stearate, talc, silicicacid, hydroxymethylcellulose, polyvinylpyrrolidone viscous paraffin andthe like.

(4) Miscellaneous Additional Components: The compositions of the presentinvention may additionally contain other adjunct componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional compatible pharmaceutically-active materials such as,e.g., antipruritics, astringents, local anesthetics or anti-inflammatoryagents, or may contain additional materials useful in physicallyformulating various dosage forms of the composition of presentinvention, such as dyes, flavoring agents, preservatives, antioxidants,opacifiers, thickening agents and stabilizers. However, such materials,when added, should not unduly interfere with the biological activitiesof the components of the compositions of the invention.

C. Colloidal Dispersion Systems: Regardless of the method by which theantisense compounds of the invention are introduced into a patient,colloidal dispersion systems may be used as delivery vehicles to enhancethe in vivo stability of the compounds and/or to target the compounds toa particular organ, tissue or cell type. Colloidal dispersion systemsinclude, but are not limited to, macromolecule complexes, nanocapsules,microspheres, beads and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, lipid: oligonucleotide complexes ofuncharacterized structure and liposomes.

A preferred colloidal dispersion system is a plurality of liposomes.Liposomes are microscopic spheres having an aqueous core surrounded byone or more outer layer(s) made up of lipids arranged in a bilayerconfiguration (see, generally, Chonn et al., Current Op. Biotech., 1995,6, 698). The therapeutic potential of liposomes as drug delivery agentswas recognized nearly thirty years ago (Sessa et al., J. Lipid Res.,1968, 9, 310). Liposomes, in some instances, may be used as cellulardelivery vehicles for bioactive agents in vitro and in vivo (Mannino etal., Biotechniques, 1988, 6, 682; Blume et al., Biochem. et Diophys.Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119.For example, it has been shown that large unilamellar vesicles (LUV) ,which range in size from 0.2-0.4 microns, can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand delivered to brain cells in a biologically active form (Fraley etal., Trends Biochem. Sci., 1981, 6, 77).

The targeting of colloidal dispersion systems, including liposomes, canbe either passive or active. Passive targeting utilizes the naturaltendency of liposomes to distribute to cells of the reticuloendothelialsystem in organs that contain sinusoidal capillaries. Active targeting,by contrast, involves modification of the liposome by coupling thereto aspecific ligand such as a viral protein coat (Morishita et al., Proc.Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474), monoclonal antibody (or asuitable binding portion thereof), sugar, glycolipid or protein (or asuitable oligopeptide fragment thereof), or by changing the compositionand/or size of the liposome in order to achieve distribution to organsand cell types other than the naturally occurring sites of localization.

The surface of the targeted colloidal dispersion system can be modifiedin a variety of ways. In the case of a liposomal targeted deliverysystem, lipid groups can be incorporated into the lipid bilayer of theliposome in order to maintain the targeting ligand in close associationwith the lipid bilayer. Various linking groups can be used for joiningthe lipid chains to the targeting ligand. The targeting ligand, whichbinds a specific cell surface molecule found predominantly on cells towhich delivery of the compounds of the invention is desired, may be, forexample, (1) a hormone, growth factor or a suitable oligopeptidefragment thereof which is bound by a specific cellular receptorpredominantly expressed by cells to which delivery is desired or (2) apolyclonal or monoclonal antibody, or a suitable fragment thereof (e.g.,Fab; F(ab')₂) which specifically binds an antigenic epitope foundpredominantly on targeted cells. Two or more bioactive agents (e.g., anantisense oligonucleotide and a conventional drug; two oligonucleotides)can be combined within, and delivered by, a single liposome. It is alsopossible to add agents to colloidal dispersion systems which enhance theintercellular stability and/or targeting of the contents thereof.

The liposomes of the invention are formed from vesicle-forming lipidswhich generally include one or more neutral or negatively chargedphospholipids, preferably one or more neutral phospholipids, usually incombination with one or more sterols, particularly cholesterol. Examplesof lipids useful in liposome production include phosphatidyl compounds,such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,sphingolipids, phosphatidylethanolamine, cerebrosides and gangliosides.Typically, the major lipid component of the liposomes is aphosphatidylcholine (PC) or PC derivative. PC derivatives with a varietyof acyl chain groups of varying chain length and degree of saturationare commercially available or may be synthesized by known techniques.For purposes of filter sterilization, less-saturated PCs are generallymore easily sized, particularly when the liposomes must be sized belowabout 0.3 microns. PCs containing saturated fatty acids with carbonchain lengths in the range of C₁₄ to C₂₂, particularly C₁₆ to C₁₈, arepreferred, particularly diacyl phosphatidylglycerols. Illustrativephospholipids include, for example, dipalmitoylphosphatidylcholine,phosphatidylcholine and distearoylphosphatidylcholine.Phosphatidylcholines with mono- and di-unsaturated fatty acids andmixtures of saturated and unsaturated fatty acids may also be used.Other suitable phospholipids include those with head groups other thancholine, such as, for example, ethanolamine, serine, glycerol andinositol. Other suitable lipids include phosphonolipids in which thefatty acids are linked to glycerol via ether linkages rather than esterlinkages. Preferred liposomes will include a sterol, e.g., cholesterol,at molar ratios of from about 0.1 to 1.0 (sterol: phospholipid).

Typically, the liposomes of the invention will contain, in their aqueousinteriors, one or more antisense oligonucleotides in an amount of fromabout 0.005 ng/mL to about 400 mg/mL, preferably from about 0.01 ng/mLto about 200 mg/mL, most preferably from about 0.1 ng/mL to about 100mg/mL, where "about" indicates ±5% of the desired concentration.

Compositions of the invention may include one or more antisensecompounds and/or other therapeutic agents entrapped within stericallystabilized liposomes. As used herein, the term "sterically stabilizedliposome" refers to a liposome comprising one or more specialized lipidsthat, when incorporated into liposomes, result in enhanced circulationlifetimes relative to liposomes lacking such specialized lipids.Examples of sterically stabilized liposomes are those in which part ofthe vesicle-forming lipid portion of the liposome (A) comprises one ormore glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (Allen et al., FEBS Letts., 1987, 223,42; Wu et al., Cancer Res., 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂ 15G, thatcontains a PEG moiety. Illum et al. (FEBS Letters, 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Letts., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. 0,445,131 BE andWO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP0,496,813 B1). Liposomes comprising a number of other lipid-polymerconjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212(both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomescomprising PEG-modified ceramide lipids are described in WO 96/10391(Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized via functional surface moieties.

A limited number of liposomes comprising nucleic acids are known in theart. Published PCT application No. WO 96/40062 to Thierry et al.discloses methods for encapsulating high molecular weight nucleic acidsin liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. disclosesprotein-bonded liposomes and asserts that the contents of such liposomesmay include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al.describes certain methods of encapsulating oligodeoxynucleotides inliposomes. WO 97/04787 to Love et al. discloses liposomes comprisingantisense oligonucleotides targeted to the raf gene. WO 97/46671 toKlimuk et al. discloses liposomes comprising antisense oligonucleotidestargeted to genes encoding ICAM-1. One or more antisense compounds ofthe invention can be formulated in a lipid: (antisense compound)!complex comprising one or more cationic lipids as disclosed in U.S. Pat.No. 5,705,385 to Bally et al. and in WO 96/40964 to Wheeler et al., orin lipoprotein-containing complexes such as are described in WO 98/00556to Kim et al.

The liposomes of the invention can be prepared by any of a variety ofknown techniques. For example, the liposomes can be formed by anyconventional technique for preparing multilamellar lipid vesicles(MLVs), i.e., by depositing one or more selected lipids on the insidewall of a suitable vessel by dissolving the lipid in chloroform,evaporating the chloroform and then adding an aqueous solution whichcomprises the agent(s) to be encapsulated to the vessel, allowing theaqueous solution to hydrate the lipid, and swirling or vortexing theresulting lipid suspension. This process yields a mixture including thedesired liposomes.

As another example, techniques used for producing large unilamellarvesicles (LUVs), such as, e.g., reverse-phase evaporation, infusionprocedures and detergent dilution, can be used to produce the liposomes.These and other methods for producing lipid vesicles are described inLiposome Technology, Volume I (Gregoriadis, Ed., CRC Press, Boca Raton,Fla., 1984). The liposomes can be in the form of steroidal lipidvesicles, stable plurilamellar vesicles (SPLVs), monophasic vesicles(MPVs) or lipid matrix carriers (LMCs) of the type disclosed in U.S.Pat. Nos. 4,588,578 and 4,610,868 (both to Fountain et al.), 4,522,803(to Lenk et al.), and 5,008,050 (to Cullis et al.). In the case of MLVs,the liposomes can be subjected to multiple (five or more) freeze-thawcycles to enhance their trapped volumes and trapping efficiencies and toprovide a more uniform interlamellar distribution of solute if desired(Mayer et al., J. Biol. Chem., 1985, 260, 802). Specific methods formaking particular oligodeoxynucleotide:liposome compositions aredescribed in U.S. Pat. No. 5,665,710 to Rahman et al.

Following their preparation, liposomes may be sized to achieve a desiredsize range and relatively narrow distribution of sized particles. Inpreferred embodiments, the liposomes have a lower range of diameters offrom about 50 to about 75 nM, most preferably about 60 nM, and an upperrange of diameters from about 75 to about 150 nM, most preferably about125 nM, where "about" indicates ±10 nM.

Several techniques are available for sizing liposomes to a desired sizerange. Sonicating a liposome suspension by either bath or probesonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization, which relies on shearing energy to fragment largeliposomes into smaller ones, is another known sizing technique in whichMLVs are recirculated through a standard emulsion homogenizer until aselected liposome size range, typically between about 0.1 and about 0.5microns, is achieved. Extrusion of liposomes through a filter ormembrane is another method for producing liposomes having a desired sizerange (see, for example, U.S. Pat. Nos. 4,737,323 to Martin et al. and5,008,050 to Cullis et al.). Other useful sizing methods are known tothose skilled in the art. In most such methods, the particle sizedistribution can be monitored by conventional laser-beam sizedetermination or other means known in the art.

Liposomes may be dehydrated, preferably under reduced pressure usingstandard freeze-drying equipment, for extended storage. Whetherdehydrated or not, the liposomes and their surrounding media can firstbe frozen in liquid nitrogen and placed under reduced pressure. Althoughthe addition of the latter freezing step makes for a longer overalldehydration process, there is less damage to the lipid vesicles, andless loss of their internal contents, when the liposomes are frozenbefore dehydration.

To ensure that the a significant portion of the liposomes will endurethe dehydration process intact, one or more protective sugars may bemade available to interact with the lipid vesicle membranes and keepthem intact as water is removed. Appropriate sugars include, but are notlimited to, trehalose, maltose, sucrose, lactose, glucose, dextran andthe like. In general, disaccharide sugars may work better thanmonosaccharide sugars, with trehalose and sucrose being particularlyeffective in most cases, but other, more complicated sugars mayalternatively be used. The amount of sugar to be used depends on thetype of sugar and the characteristics of the lipid vesicles. Personsskilled in the art can readily test various sugars and concentrations todetermine what conditions work best for a particular lipid vesiclepreparation (see, generally, Harrigan et al., Chem. Phys. Lipids, 1990,52, 139, and U.S. Pat. No. 4,880,635 to Janoff et al.). Generally, sugarconcentrations of greater than or equal to about 100 mM have been foundto result in the desired degree of protection. Once the liposomes havebeen dehydrated, they can be stored for extended periods of time untilthey are to be used. The appropriate conditions for storage will dependon the chemical composition of the lipid vesicles and their encapsulatedactive agent(s). For example, liposomes comprising heat labile agentsshould be stored under refrigerated conditions so that the potency ofthe active agent is not lost.

Two or more bioactive agents (e.g., an oligonucleotide and aconventional drug, or two or more oligonucleotides; see below) can becombined within, and delivered by, a single liposome. It is alsopossible to add agents to colloidal dispersion systems which enhance theintercellular stability and/or targeting of the contents thereof.

5. Methods of Administration of Compounds of the Invention: Theadministration of therapeutic or pharmaceutical compositions comprisingthe antisense compounds of the invention is believed to be within theskill of those in the art. In general, a patient in need of therapy orprophylaxis is administered a composition comprising one or moreantisense compounds in accordance with the invention, commonly in apharmaceutically acceptable carrier, in doses ranging from 0.01 ug to100 g per kg of body weight depending on the age of the patient and theseverity of the disorder or disease state being treated. Dosing isdependent on severity and responsiveness of the disease state to betreated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution orprevention of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual antisensecompounds, and can generally be estimated based on EC₅₀ s found to beeffective in in vitro and in vivo animal models.

A. Treatment Regimens: In the context of the invention, the term"treatment regimen" is meant to encompass therapeutic, palliative andprophylactic modalities of administration of one or more compositionscomprising one or more antisense compounds of the invention. Aparticular treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of theoligonucleotide may either be increased in the event the patient doesnot respond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disorder or diseasestate is observed, or if the disorder or disease state has been ablated.

An optimal dosing schedule is used to deliver a therapeuticallyeffective amount of the oligonucleotide being administered via aparticular mode of administration. The term "therapeutically effectiveamount," for the purposes of the invention, refers to the amount ofoligonucleotide-containing pharmaceutical composition which is effectiveto achieve an intended purpose without undesirable side effects (such astoxicity, irritation or allergic response). Although individual needsmay vary, determination of optimal ranges for effective amounts ofpharmaceutical compositions is within the skill of the art. Human dosescan be extrapolated from animal studies (Katocs et al., Chapter 27 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990). Generally, the dosage required toprovide an effective amount of a pharmaceutical composition, which canbe adjusted by one skilled in the art, will vary depending on the age,health, physical condition, weight, type and extent of the disease ordisorder of the recipient, frequency of treatment, the nature ofconcurrent therapy (if any) and the nature and scope of the desiredeffect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds.,McGraw-Hill, New York, N.Y., 1996).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the nucleic acid is administered in maintenance doses,ranging from 0.01 ug to 100 g per kg of body weight, once or more daily,to once every 20 years. For example, in the case of in individual knownor suspected of being prone to an autoimmune or inflammatory condition,prophylactic effects may be achieved by administration of preventativedoses, ranging from 0.01 ug to 100 g per kg of body weight, once or moredaily, to once every 20 years. In like fashion, an individual may bemade less susceptible to an inflammatory condition that is expected tooccur as a result of some medical treatment, e.g., graft versus hostdisease resulting from the transplantation of cells, tissue or an organinto the individual.

In another method of the invention, a first antisense oligonucleotidetargeted to a first PECAM-1 protein is used in combination with a secondantisense oligonucleotide targeted to a second PECAM-1 protein in orderto modulate such PECAM-1 proteins to a more extensive degree than can beachieved when either oligonucleotide is used individually. In variousembodiments of the invention, the first and second PECAM-1 proteinswhich are targeted by such oligonucleotides are identical, are differentPECAM-1 proteins or are different isoforms of the same PECAM-1 protein.

In some cases it may be more effective to treat a patient with acomposition comprising one or more antisense compounds of the inventionin conjunction with other, traditional therapeutic modalities in orderto increase the efficacy of a treatment regimen. In the context of theinvention, the term "treatment regimen" is meant to encompasstherapeutic, palliative and prophylactic modalities. Followingtreatment, the patient is monitored for changes in his/her condition andfor alleviation of the symptoms of the disorder or disease state. Thedosage of the therapeutic or pharmaceutical composition may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disorder or disease state is observed, or if thedisorder or disease state has been ablated.

Prophylactic modalities for high risk individuals are also encompassedby the invention. As used herein, the term "high risk individual" ismeant to refer to an individual for whom it has been determined, via,e.g., individual or family history or genetic testing, that there is asignificantly higher than normal probability of being susceptible to theonset or recurrence of a disease or disorder. As part of a treatmentregimen for a high risk individual, the individual can beprophylactically treated to prevent the onset or recurrence of thedisease or disorder. The term "prophylactically effective amount" ismeant to refer to an amount of a pharmaceutical composition whichproduces an effect observed as the prevention of the onset or recurrenceof a disease or disorder. Prophylactically effective amounts of apharmaceutical composition are typically determined by the effect theyhave compared to the effect observed when a second pharmaceuticalcomposition lacking the active agent is administered to a similarlysituated individual. The therapeutic and pharmaceutical compositions ofthe present invention may be administered in a number of ways dependingupon whether local or systemic treatment is desired and upon the area tobe treated. Typically, either oral or parenteral administration isemployed.

B. Parenteral Delivery: The term "parenteral delivery" refers to theadministration of one or more antisense compounds of the invention to ananimal in a manner other than through the digestive canal. Parenteraladministration includes intravenous (i.v.) drip, subcutaneous,intraperitoneal (i.p.) or intramuscular injection, or intrathecal orintraventricular administration. Compositions for parenteral,intrathecal or intraventricular administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. Means of preparing and administering parenteralpharmaceutical compositions are known in the art (see, e.g., Avis,Chapter 84 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990, pages 1545-1569).Parenteral means of delivery include, but are not limited to, thefollowing illustrative examples.

Intravitreal injection, for the direct delivery of drug to the vitreoushumor of a mammalian eye, is described in U.S. Pat. No. 5,591,720, thecontents of which are hereby incorporated by reference. Means ofpreparing and administering ophthalmic preparations are known in the art(see, e.g., Mullins et al., Chapter 86 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 1581-1595).

Intravenous administration of antisense oligonucleotides to variousnon-human mammals has been described by Iversen (Chapter 26 In:Antisense Research and Applications, Crooke et al., eds., CRC Press,Boca Raton, Fla., 1993, pages 461-469). Systemic delivery ofoligonucleotides to non-human mammals via intraperitoneal means has alsobeen described (Dean et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91,11766).

Intraluminal drug administration, for the direct delivery of drug to anisolated portion of a tubular organ or tissue (e.g., such as an artery,vein, ureter or urethra), may be desired for the treatment of patientswith diseases or conditions afflicting the lumen of such organs ortissues. To effect this mode of oligonucleotide administration, acatheter or cannula is surgically introduced by appropriate means. Forexample, for treatment of the left common carotid artery, a cannula isinserted thereinto via the external carotid artery. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising the antisense compounds of the invention isinfused through the cannula or catheter into the isolated segment. Afterincubation for from about 1 to about 120 minutes, during which theoligonucleotide is taken up by cells of the interior lumen of thevessel, the infusion cannula or catheter is removed and flow within thetubular organ or tissue is restored by removal of the ligatures whicheffected the isolation of a segment thereof (Morishita et al., Proc.Natl. Acad. Sci. U.S.A., 1993, 90, 8474). Antisense oligonucleotides mayalso be combined with a biocompatible matrix, such as a hydrogelmaterial, and applied directly to vascular tissue in vivo (Rosenberg etal., U.S. Pat. No. 5,593,974, issued Jan. 14, 1997).

Intraventricular drug administration, for the direct delivery of drug tothe brain of a patient, may be desired for the treatment of patientswith diseases or conditions afflicting the brain. To effect this mode ofoligonucleotide administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region (Zimmet al., Cancer Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72(11Suppl., 3416). The pump is used to inject the oligonucleotides andallows precise dosage adjustments and variation in dosage schedules withthe aid of an external programming device. The reservoir capacity of thepump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h.Depending on the frequency of administration, ranging from daily tomonthly, and the dose of drug to be administered, ranging from 0.01 ugto 100 g per kg of body weight, the pump reservoir may be refilled at3-10 week intervals. Refilling of the pump is accomplished bypercutaneous puncture of the pump's self-sealing septum.

Intrathecal drug administration, for the introduction of a drug into thespinal column of a patient may be desired for the treatment of patientswith diseases of the central nervous system (CNS). To effect this routeof oligonucleotide administration, a silicon catheter is surgicallyimplanted into the L3-4 lumbar spinal interspace of a human patient, andis connected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region (Luer and Hatton, The Annals ofPharmacotherapy, 1993, 27, 912, 1993; Ettinger et al. Cancer, 1978, 41,1270; Yaida et al., Regul. Pept., 1985, 59, 193). The pump is used toinject the oligonucleotides and allows precise dosage adjustments andvariations in dose schedules with the aid of an external programmingdevice. The reservoir capacity of the pump is 18-20 mL, and infusionrates may vary from 0.1 mL/h to 1 mL/h. Depending on the frequency ofdrug administration, ranging from daily to monthly, and dosage of drugto be administered, ranging from 0.01 ug to 100 g per kg of body weight,the pump reservoir may be refilled at 3-10 week intervals. Refilling ofthe pump is accomplished by a single percutaneous puncture to theself-sealing septum of the pump. The distribution, stability andpharmacokinetics of oligonucleotides within the CNS are followedaccording to known methods (Whitesell et al., Proc. Natl. Acad. Sci.U.S.A., 1993, 90, 4665).

To effect delivery of oligonucleotides to areas other than the brain orspinal column via this method, the silicon catheter is configured toconnect the subcutaneous infusion pump to, e.g., the hepatic artery, fordelivery to the liver (Kemeny et al., Cancer, 1993, 71, 1964). Infusionpumps may also be used to effect systemic delivery of oligonucleotides(Ewel et al., Cancer Res., 1992, 52, 3005; Rubenstein et al., J. Surg.Oncol., 1996, 62, 194).

Epidermal and Transdernal Delivery, in which pharmaceutical compositionscontaining drugs are applied topically, can be used to administer drugsto be absorbed by the local dermis or for further penetration andabsorption by underlying tissues, respectively. Means of preparing andadministering medications topically are known in the art (see, e.g.,Block, Chapter 87 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1596-1609).

Vaginal Delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. This mode of administration may be preferred for antisenseoligonucleotides targeted to pathogenic organisms for which the vaginais the usual habitat, e.g., Trichomonas vaginalis. In anotherembodiment, antisense oligonucleotides to genes encoding sperm-specificantibodies can be delivered by this mode of administration in order toincrease the probability of conception and subsequent pregnancy. Vaginalsuppositories (Block, Chapter 87 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 1609-1614) or topical ointments can be used to effect thismode of delivery.

Intravesical Delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. However, the method requires urethral catheterization ofthe patient and a skilled staff. Nevertheless, this mode ofadministration may be preferred for antisense oligonucleotides targetedto pathogenic organisms, such as T. vaginalis, which may invade theurogenital tract.

C. Alimentary Delivery: The term "alimentary delivery" refers to theadministration, directly or otherwise, to a portion of the alimentarycanal of an animal, of a composition comprising one or more of theantisense compounds of the invention. The term "alimentary canal" refersto the tubular passage in an animal that functions in the digestion andabsorption of food and the elimination of food residue, which runs fromthe mouth to the anus, and any and all of its portions or segments,e.g., the oral cavity, the esophagus, the stomach, the small and largeintestines and the colon, as well as compound portions thereof such as,e.g., the gastro-intestinal tract. Thus, the term "alimentary delivery"encompasses several routes of administration including, but not limitedto, oral, rectal, endoscopic and sublingual/buccal administration.Compositions for alimentary delivery may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Means of preparing and administering oral pharmaceuticalcompositions are known in the art (see, e.g--, Block, Chapter 87;Rudnic, Chapter 89; Porter, Chapter 90; and Longer, Chapter 91, In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1596-1614, 1633-1665, 1666-1675and 1676-1693, respectively). Preferred compositions for the alimentarydelivery of the antisense compounds of the invention are described inco-pending U.S. patent application Ser. No. 08/886,829 to Teng et al.,filed Jul. 1, 1997, the entire disclosure of which is herebyincorporated by reference.

Buccal/Sublingual Administration: Delivery of a drug via the oral mucosahas several desirable features, including, in many instances, a morerapid rise in plasma concentration of the drug than via oral delivery(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).Furthermore, because venous drainage from the mouth is to the superiorvena cava, this route also bypasses rapid first-pass metabolism by theliver. Both features contribute to the sublingual route being the modeof choice for nitroglycerin (Benet et al., Chapter 1 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996, page 7).

Endoscopic Administration: Endoscopy can be used for drug deliverydirectly to an interior portion of the alimentary tract. For example,endoscopic retrograde cystopancreatography (ERCP) takes advantage ofextended gastroscopy and permits selective access to the biliary tractand the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992,19(10 Suppl.), 1591). Pharmaceutical compositions, including liposomalformulations, can be delivered directly into portions of the alimentarycanal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995,12, 149) or the gastric submucosa (Akamo et al., Japanese J. CancerRes., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoueet al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feedingdevices (Pennington et al., Aliment. Pharmacol. Ther., 1995, 9, 471) canalso be used for direct alimentary delivery of pharmaceuticalcompositions.

Rectal Administration: Drugs administered by the oral route can often bealternatively administered by the lower enteral route, i.e., through theanal portal into the rectum or lower intestine. Rectal suppositories,retention enemas or rectal catheters can be used for this purpose andmay be preferred when patient compliance might otherwise be difficult toachieve (e.g., in pediatric and geriatric applications, or when thepatient is vomiting or unconscious). Rectal administration may result inmore prompt and higher blood levels than the oral route, but theconverse may be true as well (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711). Because about 50% of the drug that isabsorbed from the rectum will bypass the liver, administration by thisroute significantly reduces the potential for first-pass metabolism(Benet et al., Chapter 1 In: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, NewYork, N.Y., 1996).

Oral Administration: The preferred method of administration is oraldelivery, which is typically the most convenient route for access to thesystemic circulation. Absorption from the alimentary canal is governedby factors that are generally applicable, e.g., surface area forabsorption, blood flow to the site of absorption, the physical state ofthe drug and its concentration at the site of absorption (Benet et al.,Chapter 1 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 5-7). Orally administered compositions comprisingcertain oligonucleotides are known in the art (see, for example, U.S.Pat. No. 5,591,721 to Agrawal et al.). Preferred compositions for theoral delivery of the antisense compounds of the invention are describedin co-pending U.S. patent application Ser. No. 08/886,829 to Teng etal., filed Jul. 1, 1997, incorporated herein by reference.

EXAMPLES

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

Example 1

Synthesis of Oligonucleotides

A. General Synthetic Techniques: Oligonucleotides were synthesized on anautomated DNA synthesizer using standard phosphoramidite chemistry withoxidation using iodine. β-Cyanoethyldiisopropyl phosphoramidites werepurchased from Applied Biosystems (Foster City, Calif.). Forphosphorothioate oligonucleotides, the standard oxidation bottle wasreplaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxidein acetonitrile for the stepwise thiation of the phosphite linkages.

The synthesis of 2'-O-methyl- (a.k.a. 2'-methoxy-) phosphorothioateoligonucleotides is according to the procedures set forth abovesubstituting 2'-O-methyl β-cyanoethyldiisopropyl phosphoramidites(Chemgenes, Needham, Mass.) for standard phosphoramidites and increasingthe wait cycle after the pulse delivery of tetrazole and base to 360seconds.

Similarly, 2'-O-propyl- (a.k.a 2'-propoxy-) phosphorothioateoligonucleotides are prepared by slight modifications of this procedureand essentially according to procedures disclosed in U.S. patentapplication Ser. No. 08/383,666, filed Feb. 3, 1995, which is assignedto the same assignee as the instant application.

The 2'-fluoro-phosphorothioate antisense compounds of the invention aresynthesized using 5'-dimethoxytrityl-3'-phosphoramidites and prepared asdisclosed in U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both ofwhich are assigned to the same assignee as the instant application. The2'-fluoro-oligonucleotides were prepared using phosphoramidite chemistryand a slight modification of the standard DNA synthesis protocol (i.e.,deprotection was effected using methanolic ammonia at room temperature).

The 2'-methoxyethyl oligonucleotides were synthesized essentiallyaccording to the methods of Martin et al. (Helv. Chim. Acta, 1995, 78,486). For ease of synthesis, the 3' nucleotide of the 2'-methoxyethyloligonucleotides was a deoxynucleotide, and 2'--0--CH₂ CH₂OCH₃,cytosines were 5-methyl cytosines, which were synthesized accordingto the procedures described below.

PNA antisense analogs are prepared essentially as described in U.S. Pat.Nos. 5,539,082 and 5,539,083, both of which (1) issued Jul. 23, 1996,and (2) are assigned to the same assignee as the instant application.

B. 5-Methyl-2'-Methoxyethoxy-Cytosine: Oligonucleotides having5-methyl-2'-methoxyethoxy-cytosine residues are prepared as follows.

(i) 2,2'-Anhydro 1-(β-D-arabinofuranosyl)-5-methyluridine!:5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279M), diphenylcarbonate (90.0 g, 0.420M) andsodium bicarbonate (2.0 g, 0.024M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

(ii) 2'-O-Methoxyethyl-5-methyluridine: 2,2'-Anhydro-5-methyluridine(195 g, 0.81M), tris(2-methoxyethyl)borate (231 g, 0.98M) and2-methoxyethanol (1.2L) were added to a 2L stainless steel pressurevessel and placed in a pre-heated oil bath at 160° C. After heating for48 hours at 155-160° C., the vessel was opened and the solutionevaporated to dryness and triturated with MeOH (200 mL). The residue wassuspended in hot acetone (1L). The insoluble salts were filtered, washedwith acetone (150 mL) and the filtrate evaporated. The residue (280 g)was dissolved in CH₃ CN (600 mL) and evaporated. A silica gel column (3kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3) containing 0.5% Et₃ NH.The residue was dissolved in CH₂ Cl₂ (250 mL) and adsorbed onto silica(150 g) prior to loading onto the column. The product was eluted withthe packing solvent to give 160 g (63%) of product.

(iii) 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506M) was co-evaporated withpyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). Afirst aliquot of dimethoxytrityl chloride (94.3 g, 0.278M) was added andthe mixture stirred at room temperature for one hour. A second aliquotof dimethoxytrityl chloride (94.3 g, 0.278N) was added and the reactionstirred for an additional one hour. Methanol (170 mL) was then added tostop the reaction. HPLC showed the presence of approximately 70%product. The solvent was evaporated and triturated with OH₃ ON (200 mL).The residue was dissolved in CHCl₃ (1.5L) and extracted with 2×500 mL ofsaturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase wasdried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

(iv) 3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167M) ,DNF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DNF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approximately90% product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%).

(v)3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine:A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44M) was added to a solution of triazole (90 g, 1.3M) in CH₃ CN (1L),cooled to -5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

(vi) 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: A solutionof3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. Methanol (400 mL) saturated with NH₃ gas was added and thevessel heated to 100° C. for 2 hours (thin layer chromatography, tlc,showed complete conversion). The vessel contents were evaporated todryness and the residue was dissolved in EtOAc (500 mL) and washed oncewith saturated NaCl (200 mL). The organics were dried over sodiumsulfate and the solvent was evaporated to give 85 g (95%) of the titlecompound.

(vii) N⁴-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165M) wasadded with stirring. After stirring for 3 hours, tlc showed the reactionto be approximately 95% complete. The solvent was evaporated and theresidue azeotroped with MeOH (200 mL). The residue was dissolved inCHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

(viii) N⁴-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite:N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10M) was dissolved in CH₂ Cl₂ (1L). Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (tlc showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (3×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂ Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAcHexane (3:1) as theeluting solvent. The pure fractions were combined to give 90.6 g (87%)of the title compound.

C. Purification: After cleavage from the controlled pore glass column(Applied Biosystems) and deblocking in concentrated ammonium hydroxideat 55° C. for 18 hours, the oligonucleotides were purified byprecipitation twice out of 0.5M NaCl with 2.5 volumes ethanol.Analytical gel electrophoresis was accomplished in 20% acrylamide, 8Murea, 45 mM Tris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothicate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2

Nucleotide Sequences of Oligonucleotides Targeted to PECAM-1

A. Oligonucleotides Targeted to Nucleic Acids Encoding Human PECAM-1:Table 1 lists the nucleotide sequences of a set of oligonucleotidesdesigned to specifically hybridize to human PECAM-1 mRNAs and theircorresponding ISIS and SEQ ID numbers. The nucleotide co-ordinates ofthe target gene and gene target regions are also included. Thenucleotide co-ordinates are derived from GenBank Accession No. M28526,locus name "HUMPECAM1" (see also Newman et al., Science, 1990, 247,1219; SEQ ID NO: 1). The abbreviations for gene target regions are asfollows: 5'-UTR, 5' untranslated region; AUG, translation initiationregion; ORF, open reading frame; stop, translation termination region;3'-UTR, 3' untranslated region. The location of the target sequencescomplementary to those of the oligonucleotides is shown in FIG. 1.

The nucleotides of the oligonucleotides whose sequences are presented inTable 1 are connected by phosphorothioate linkages and are unmodified atthe 2' position (i.e., 2'-deoxy). After studies involving this initialset of oligonucleotides were conducted, a second group ofoligonucleotides comprising additional chemical modifications wasprepared. These "second generation" oligonucleotides, some of which arederived from the initial phosphorothioate oligonucleotides, aredescribed in Table 2.

                                      TABLE 1    __________________________________________________________________________    Nucleotide Sequences of Phosphorothioate    Oligonucleotides Targeted to Human PECAM-1                        SEQ TARGET GENE                                      GENE    ISIS NUCLEOTIDE SEQUENCE                        ID  NUCLEOTIDE                                      TARGET    > 3')(5'         NO             CO-ORDINATES.sup.1                            REGION    __________________________________________________________________________    8988 GCTCTGGTCACTTCTCCCGG                        2   0006-0025 5'-UTR    8989 GAGAAACCCGCCCTGTGAAA                        3   0036-0055 5'-UTR    8990 TCGGGCCATGACTCGCTCAG                        4   0084-0103 5'-UTR    8991 CTGCATCCTGAGAGTGAAGA                        5   0128-0147 AUG    8992 TGCACCGTCCAGTCCGGCAG                        6   0265-0284 ORF    8993 CCATGTCTGTTGTGGGCCAC                        7   0976-0995 ORF    8994 GTGCATCTGGCCTTGCTGTC                        8   2358-2377 stop    8995 GGAGCAGGGCAGGTTCATAA                        9   2449-2468 3'-UTR    8996 AACCGGCAGCTTAGCCTGAG                        10  2487-2506 3'-UTR    11591         GAGCAGGGCAGGTTCATAAA                        11  2448-2467 3'-UTR    11593         AGCTTAGCCTGAGGAATTGC                        12  2480-2499 3'-UTR    11594         CCATCAAGGGAGCCTTCCGT                        13  2332-2351 ORF    11595         CCAGGGATGTGCATCTGGCC                        14  2367-2385 3'-UTR    11914         GACAGCAAGGCCAGATGCAC                        15  sense control                                      8994    11915         TGCGTAGCTCGCGTCTGTCT                        16  scrambled control                                      8994    __________________________________________________________________________     .sup.1 Coordinates from Genbank Accession No. M28526, locus name     "HUMPECAM1", SEQ ID NO: 1.     (N.B.: SEQ ID NOs: 17, 18 = phosphodiester primers for PCR of human     PECAM1)

                                      TABLE 2    __________________________________________________________________________    Nucleotide Sequences of "Second Generation"    Oligonucleotides Targeted to Human PECAM-1                                 SEQ TARGET    ISIS NUCLEOTIDE SEQUENCE (5'→3')                                 ID  REGION    NO   AND CHEMICAL MODIFICATIONS.sup.1                                 NO  (BASES).sup.2    __________________________________________________________________________     8994         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    ISIS 8994-derived gapmers:    12466          GoToGoCoAoToCsTsGsGsCsCsTsTsGoCoToGoToC                                 8   stop                                     2358-                                     2377    12467         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17555         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17556         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17557         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17559         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17560         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    ISIS 8994 and 8995 derived hemimers:    17566         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17651         GsTsGsCsAsTsCsTsGsGsCsCsTsTsGsCsTsGsTsC                                 8   stop                                     2358-                                     2377    17567         GsGsAsGsCsAsGsGsGsCsAsGsGsTsTsCsAsTsAsA                                 9   3'-UTR                                     2449-                                     2468    Direct "Second-Generation" Compounds:    17561         TsCsCsTsTsCsCsAsGsGsGsAsTsGsTsGsCsAsTsC                                 19  3'UTR                                     2371-                                     2390    17562         TsGsAsAsAsTsAsCsAsGsGsGsAsTsTsAsTsCsTsG                                 20  3'UTR                                     2411-                                     2430    17563         TsGsGsGsAsGsCsAsGsGsGsCsAsGsGsTsTsCsAsT                                 21  3'UTR                                     2451-                                     2470    17564         TsGsGsCsCsTsTsGsCsTsGsTsCsTsAsAsGsTsTsC                                 22  stop                                     2351-                                     2370    17565         AsCsAsGsCsTsTsTsCsCsGsGsAsCsTsTsCsAsCsT                                 23  ORF                                     2281-                                     2300    17568         CsTsAsGsAsGsTsAsTsCsTsGsCsTsTsTsCsC sAsC                                 24  ORF                                     2311-                                     2330    17569         CsAsCsTsTsTsGsTsCsAsAsTsAsCsCsTsGsCsAsG                                 25  ORF                                     1301-                                     1320    17570         AsCsTsCsTsCsTsGsTsGsCsTsCsTsTsCsAsTsGsG                                 26  ORF                                     0401-                                     0420    14391         GsAsCsGsCsAsTsCsGsCsGsCsCsTsAsCsAsTsCsG                                 27  ICAM-1                                     scrambled                                     control    17966         TsCsCsTsTsCsCsTsGsGsGsAsGsGsTsAsCsAsTsC                                 28  17561                                     3-base                                     mismatch    17970         CsTsTsCsGsAsGsAsCsGsCsTsGsCsTsCsAsTsGsT                                 29  17561                                     scrambled                                     control    __________________________________________________________________________     .sup.1 Emboldened residues, 2methoxyethoxy-residues (others are 2deoxy-)     underlined C ("C" or "C") residues, 5methyl-cytosines; "o", phosphodieste     linkage; "s", phosphorothioate linkage.     .sup.2 Base coordinates from Genbank Accession No. M28526, locus name     "HUMPECAM1", SEQ ID NO: 1.

B. Oligonucleotides Targeted to Nucleic Acids Encoding Murine PECAM-1:Tables 3 and 4 describe the nucleotide sequences and chemistries of aset of oligonucleotides designed to specifically hybridize to murinePECAM-1-encoding mRNAs and their corresponding ISIS and SEQ ID numbers.The nucleotide co-ordinates of the target gene and gene target regionsare also included. The nucleotide co-ordinates are derived from GenBankaccession No. L06039, locus name "MUSPECAM1" (see also FIG. 1 of Xie etal., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 5569; SEQ ID NO: 30).

                                      TABLE 3    __________________________________________________________________________    Nucleotide Sequences of Phosphorothioate    Oligonucleotides Targeted to Murine PECAM-1                         SEQ                            TARGET GENE                                     GENE    ISIS NUCLEOTIDE SEQUENCE                         ID NUCLEOTIDE                                     TARGET    > 3')(5'         NO              CO-ORDINATES.sup.1                            REGION    __________________________________________________________________________    13983         GAAGCCTTCCGTTCTAGAGTAT                         31 2246-2267                                     ORF    13984         CGCTGGTGCTCTATGCAAGCCT                         32 0121-0142                                     ORF    13985         GTCTTCCGGCCATCCTCAGG                         33 0005-0024                                     5'-UTR    13986         TGCAGACTGAAGCACTCAGC                         34 0032-0051                                     5'-UTR    13987         CATCTTTGCTGCCGACTGAG                         35 0082-0101                                     AUG    13988         GATGTCCACAAGGCACTCCA                         36 0232-0251                                     ORF    13989         GGATACGCCATGCACCTTCA                         37 0442-0461                                     ORF    13990         CAAAACGCTTGGGTGTCATT                         38 1716-1735                                     ORF    13991         CCGCAATGAGCCCTTTCTTC                         39 1862-1881                                     ORF    13992         CCAATGACAACCACCGCAAT                         40 1875-1894                                     ORF    13993         GTAATGGCTGTTGGCTTCCA                         41 2035-2054                                     ORF    13994         GTCTCTGTGGCTCTCGTTCC                         42 2178-2197                                     ORF    13995         TGCACTGCCTTGACTGTCTT                         43 2281-2300                                     stop    13996         GAATCGGCTGCTCTTCTCGG                         44 2325-2344                                     3'-UTR    13997         GGATTACTGCTTTCGGTGGG                         45 2386-2405                                     3'-UTR    13998         CCCAACATGAACAAGGCAGC                         46 2436-2455                                     3'-UTR    13999         CACAAGGAAGATAGGTCAGG                         47 2491-2510                                     3'-UTR    14447         CCGACATCAGCGTTCTCTCT                         48 13991 control                                     scrambled    14448         AGCACGTCCTTGTTCGTTCT                         49 13995 control                                     scrambled    14449         GTAGAGCCTACGTACCTCCA                         50 13989 control                                     scrambled    __________________________________________________________________________     .sup.1 Coordinates from Genbank Accession No. L06039, locus name     "MUSPECAMI", SEQ ID NO: 30.     (N.B.: SEQ ID NOs: 51, 52 = phosphodiester primers for PCR of mouse PECAM     (ISIS 14244, 14245, respectively))

                                      TABLE 4    __________________________________________________________________________    Nucleotide Sequences of "Second Generation"    Oligonucleotides Targeted to Murine PECAM-1                                SEQ  TARGET    ISIS NUCLEOTIDE SEQUENCE (5'→3')                                ID   REGION    NO   AND CHEMICAL MODIFICATIONS.sup.1                                NO   (BASES).sup.2    __________________________________________________________________________    13989         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17049         GsGsAsTsAsCsGsCsCsAs TsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17050         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17051         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17052         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17053         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    17054         GsGsAsTsAsCsGsCsCsAsTsGsCsAsCsCsTsTsCsA                                37   ORF                                     0442-                                     0461    20380         GoGsAsTsAsCsGsCsCsAsTsGsCoAoCoCoToToCoA37                                ORF                                     0442-                                     0461    18539         GsAsCsAsTsCsGsCsGsAsTsAsCsCsTsCsTsAsC                                53   13989                                     scrambled                                     control    20381         GoAsGsCsAsTsCsGsCsGsAsTsAoCoCoToCoToAoC                                53   13989                                     scrambled                                     control    __________________________________________________________________________     .sup.1 Emboldened residues, 2methoxyethoxy- residues (others are 2deoxy-)     including "C" residues, 5methyl-cytosines; "o", phosphodiester linkage;     "s", phosphorothioate linkage.     .sup.2 Base coordinates from Genbank Accession No. L06039, locus name     "MUSPECAMI", SEQ ID NO: 30.

Example 3

Assays for Oligonucleotide-Mediated Inhibition of PECAM-1 mRNAExpression in HUVEC Cells

A. General Techniques: In order to evaluate the activity of potentialhuman PECAM-1-modulating oligonucleotides, human umbilical veinendothelial cells (HUVEC) from Clonetics Corporation (Walkersville, Md.;alternatively, ATCC CRL-1730 from the American Type Culture Collection,Rockville, Md., can be used) were grown and treated witholigonucleotides or control solutions as detailed below. Foroligonucleotides targeting the murine PECAM-1 gene,oligonucleotide-treated cells were of the bEND.3 endothelial cell line(gift of Dr. Werner Risau; see Montesano et al., Cell, 1990, 62, 435,and Stepkowski et al., J. Tmmunol., 1994, 153, 5336). After harvesting,cellular extracts were prepared and examined for specificPECAM-1-encoding mRNA levels or PECAM-1 protein levels (e.g., Northernor flow cytometry assays, respectively). In all cases, "% expression"refers to the amount of PECAM-1-specific signal in anoligonucleotide-treated cell relative to an untreated cell (or a celltreated with a control solution that lacks oligonucleotide), and "%inhibition" is calculated as

    100%-% Expression=% Inhibition.

B. RNA Assays: The mRNA expression of each PECAM-1 protein wasdetermined by using a nucleic acid probe specifically hybridizablethereto in "Northern" assays. Nucleic acid probes specific for human andmurine PECAM-1 are described in Examples 4-6. The probes wereradiolabelled by means well known in the art (see, e.g., Short Protocolsin Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons,New York, 1992, pages 3-11 to 2-3-44 and 4-17 to 4-18; Ruth, Chapter 6in: Methods in Molecular Biology, Vol. 26: Protocols for OligonucleotideConjugates, Agrawal, ed., Humana Press Inc., Totowa, N.J., 1994, pages167-185; and Chapter 10 In: Molecular Cloning: A Laboratory Manual, 2ndEd., Sambrook et al., eds., pages 10.1-10.70). The blots were strippedand reprobed with a ³² P-labeled glyceraldehyde 3-phosphatedehydrogenase (G3PDH) probe (Clontech Laboratories, Inc., Palo Alto,Calif.) in order to confirm equal loading of RNA and to allow the levelsof PECAM-1 transcripts to be normalized with regard to the G3PDHsignals.

HUVEC (human) or bEND.3 (murine) cells were grown in, respectively, EGM(Clonetics, Walkersville, Md.) or high-glucose DMEM (GIBCO-BRL LifeTechnologies, Gaithersburg, Md.) media containing 10% fetal bovine serum(FBS) in T-75 flasks until 80-90% confluent. At this time, the cellswere washed 3× with 10 mL of Opti-MEM media (GIBCO-BRL). Then, 5 mL ofOpti-MEM media containing 10 (HUVEC) or 15 (bEND.3) ug/mL LIPOFECTIN®(i.e., 1:1 (w/w) DOTMA/DOPE, where DOTMA=N-1-(2,3-dioleyoxy)propyl!-N,N,N-trimethylammonium chloride andDOPE=dioleoyl phosphatidylethanolamine; GIBCO-ERL) and an appropriateamount of oligonucleotide were added to the cells (the time of additionof oligonucleotide is t=0 h in the experiments described herein). As acontrol, cells were treated with LIPOFECTIN® without oligonucleotideunder the same conditions and for the same times as theoligonucleotide-treated samples. After 4 hours at 37° C. (t=4 h), themedium was replaced with fresh EGM (HUVEC) or high glucose DMEM (bEND.3)media containing 10% FES. The cells were typically allowed to recoverfor 2 hours. Total cellular RNA was then extracted in guanidinium,subjected to gel electrophoresis and transferred to a filter accordingto techniques known in the art (see, e.g., Chapter 7 In: MolecularCloning: A Laboratory Manual, 2nd Ed., Sambrook et al., eds., pages7.1-7.87, and Short Protocols in Molecular Biology, 2nd Ed., Ausubel etal., eds., John Wiley & Sons, New York, 1992, pages 2-24 to 2-30 and4-14 to 4-29).

Following RNA transfer, filters were typically hybridized overnight to aprobe specific for the particular PECAM-1-encoding gene of interest inhybridization buffer (QUIKHYB™ hybridization solution, Stratagene, LaJolla, Calif.). This was followed by two washes in 2×SSC, 0.1% SDS atroom temperature (˜24° C.) for 15 minutes and one wash in 0.1% SSC, 0.1%SDS at 60° C. for 30 minutes. Hybridizing bands were visualized byexposure to X-OMAT AR film and quantitated using a PHOSPHORIMAGER®essentially according to the manufacturer's instructions (MolecularDynamics, Sunnyvale, Calif.). Although quantitation via aPHOSPHORIMAGER® or a comparable instrument is a preferred means ofmeasuring RNA levels, the results of these "Northern" assays could bedetermined by other means known in the art.

C. Protein Assays: HUVEC or bEND.3 cells were grown in 12 well platesand treated with oligonucleotides as described above, except that cellswere generally allowed to recover overnight (e.g., generally about 18hours) before protein extracts were prepared. Cells were washed andreleased by treatment with trypsin (GIBCO-BRL) or EDTA. Specifically,H-VEC cells were typically released by trypsin treatment, although EDTAtreatment worked as well, whereas bEND.3 cells are preferentiallyreleased by treatment with 2 mM EDTA in Ca⁺⁺ - and Mg⁺⁺ -free 1× PBSbuffer (HESS; GIBCO-BRL). Cells were stained with an appropriatefluorescently labeled primary antibody that specifically recognizes thePECAM-1 protein under examination and the amount of each PECAM-1 proteinwas determined by using fluorescence-activated cell sorting (FACS®)techniques (see, e.g., U.S. Pat. Nos. 4,727,020 to Recktenwald,5,223,398 to Kortright et al., and 5,556,764 to Sizto et al.). Thefluorescently labelled primary antibodies specific for each PECAM-1protein are described in the appropriate Examples. Alternatively,unlabelled primary antibodies to PECAM-1 (some are described in theExamples) can be used and detected by the use of fluorescently-labelledsecondary (i.e., specific for the primary antibody) antibodies.

In alternative methods for measuring PECAM-1 levels, cell lysates andprotein extracts are electrophoresed (SDS-PAGE), transferred tonitrocellulose filters and detected by means known in the art (see,e.g., Chapter 18 In: Molecular Cloning: A Laboratory Manual, 2nd Ed.,Sambrook et al., eds., pages 18.34, 18.47-18.54 and 18.60-18.75)).Unlabelled primary antibodies to PECAM-1 can be used and detected bymeans well known in the art including, for example, detection of theprimary antibody by a secondary antibody that binds the primary antibody(see, e.g., Short Protocols in Molecular Biology, 2nd Ed., Ausubel etal., eds., John Wiley & Sons, New York, 1992, pages 10-33 to 10-35; andChapter 18 In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrooket al., eds., pages 18.1-18.75 and 18.86-18.88) and quantitated usingother antibody-based assays known in the art. Such antibody-based assaysinclude, but are not limited to, ELISA assays, Western assays, and thelike (see, for example, U.S. Pat. Nos. 4,879,219 to Wands et al. and4,837,167 to Schoemaker et al., and Short Protocols in MolecularBiology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York,1992, pages 11-5 to 11-17). PECAM-1 activity can be measured inappropriate cell adhesion assays known in the art (see, for example,Albelda et al., J. Cell Biol., 1991, 114, 1059; DeLisser et al., J.Biol. Chem., 1993, 268, 16307; and Muller et al., J. Exp. Med., 1992,175, 1401).

Example 4

Antisense-Mediated Inhibition of Human PECAM-1 Expression byPhosphorothioate Oligonucleotides

A. Human PECAM-l-Specific Probes: In initial screenings of a set ofoligonucleotides derived from the human PECAM-1 sequence (Table 1) forbiological activity, a radiolabelled PECAM-1-specific PCR product wasprepared using RNA from HUVEC cells and a pair of phosphodiesteroligonucleotides,

5'-TCCGATGTCAAGCTAGGATCAT (SEQ ID NO: 17); and

5'-GGCATGGGAATGGCAATTATCT (SEQ ID NO: 18),

as primers in PCR reactions using a PRIME-A-GENE labeling kit (Promega,Madison, Wis.). The resultant ³² P-cytosine-radiolabelled 844-bp producthybridizes to the open reading frame of human PECAM-1 (nucleotides752-1596 of Genbank Accession No. M28526, locus name "HUMPECAM1")Alternatively, however, other PCR probes can be prepared, or one or moreof the oligonucleotides of Tables 1 or 2 can be detectably labeled andused as a human PECAM-1-specific probe using methods known in the art.

B. Activities of PECAM-1 Oligonucleotides: In an initial screen foractive compounds capable of modulating PECAM-1 expression, HUVEC cellswere treated with 100 nM of a series of PECAM-1-specificphosphorothioate oligonucleotides (ISIS 8988-8996, SEQ ID NOs: 2-10) andPECAM-1 mRNA levels were determined by Northern analysis. The data fromthe screening (Table 5, Experiment 1) indicate the following results.Oligonucleotides showing activity in this assay, as reflected by levelsof inhibition from ≧about 35% to about 100% of PECAM-1 mRNA levels,where "about" indicates ±5%, include ISIS 8988, 8991, 8992, 8993, 8994,8995 and 8996 (SEQ ID NOs: 2, 5, 6, 7, 8, 9 and 10, respectively). Theseoligonucleotides are thus preferred embodiments of the invention formodulating PECAM-1 expression. Oligonucleotides showing levels ofinhibition of from ≧about 50% to about 100% of PECAM-1 mRNAs in thisassay, where "about" indicates ±5%, include ISIS 8994, 8995 and 8996(SEQ ID NOs: 8, 9 and 10, respectively). These oligonucleotides are thusmore preferred embodiments of the invention for modulating PECAM-1expression.

Screening of a second set of phosphorothioate oligonucleotides (Table 5,Experiment 2) yielded additional antisense sequences against humanPECAM-1. Specifically, oligonucleotides showing activity in this assay,as reflected by levels of inhibition from ≧about 50% to about 100% ofPECAM-1 mRNA levels, where "about" indicates ±5%, include ISIS 11591,11593, 11594 and 11595 (SEQ ID NOs: 11, 12, 13 and 14, respectively);these oligonucleotides are thus preferred embodiments of the inventionfor modulating PECAM-1 expression. Oligonucleotides showing levels ofinhibition of from ≧about 85% to about 100% of PECAM-1 mRNAs in thisassay, where "about" indicates ±5%, include ISIS 11594 and 11595 (SEQ IDNOs: 13 and 14, respectively). These oligonucleotides are thus morepreferred embodiments of the invention for modulating PECAM-1expression.

Among the phosphorothioate oligonucleotides initially screened, the mostpreferred embodiments of the invention are those showing levels ofinhibition of from ≧about 55% to about 100% of PECAM-1 mRNAs in thisassay, where "about" indicates ±5%, i.e., are ISIS 8994, 8996, 11591,11593, 11594 and 11595 (SEQ ID NOs: 8, 10, 11, 12, 13 and 14,respectively).

C. Sequence Specificity: The specificity of modulation of PECAM-1 by theantisense oligonucleotides was confirmed using ISIS 8994 and a set ofcontrol oligonucleotides having sequences that are either sense (i.e.,reverse complement) or scrambled versions of that of the activeoligonucleotide. Specifically, ISIS 11914 (SEQ ID NO: 15) is a "sense"control, and ISIS 11915 (SEQ ID NO: 16) is a "scrambled" control, forISIS 8994. HUVEC cells were treated with these oligonucleotides at 50 nMand evaluated by Northern analysis as above. The results (Table 6)demonstrate that, although the active oligonucleotide ISIS 8994 caused90% inhibition of PECAM-1 mRNA under these conditions, treatment ofcells with the control oligonucleotides had little effect on PECAM-1mRNA expression (i.e., 14% inhibition for ISIS 11914 and 26% for ISIS11915). Thus, the PECAM-1-modulating activity of the active antisensecompounds is sequence-specific.

D. Time Course: The time course of inhibition of PECAM-1 mRNA expressionin H-VEC cells following treatment with 100 nM of ISIS 8994 or 8996 isshown in Table 7. After 4 hours of treatment with ISIS 8994, the levelof inhibition of PECAM-1 was greater than about 85% (t=4 h), rose toabout 98% inhibition at t=6 h, and subsequently remained at greater thanor equal to about 95%, where "about" indicates ±5%, until at least t=12h. A similar pattern of temporal modulation of human PECAM-1 was seenfor ISIS 8996.

E. Protein Levels: In order to assess the correlation between PECAM-1mRNA and protein levels, experiments were performed in which HUVEC cellswere treated with 50 nM of various oligonucleotides and samples weretaken at t=4 h, 24 h and 48 h. The samples were split and (1) levels ofPECAM-1 protein were determined by FACS using a monoclonal antibody toPECAM-1 (CD31) conjugated to phycoerythrin (PE) from Becton-Dickinson(Franklin Lakes, N.J.) and (2) PECAM-1 mRNA levels were determined as inthe previous Examples. FACS results were similar whether cells werelysed with trypsin or EDTA and whether PE- or FITC- (fluoresceinisothiocyanate) labelled antibodies to human PECAM-1 were used. PE- andFITC- labelled antibodies to human PECAM-1 are available from, forexample, Becton-Dickinson (Franklin Lakes, N.J.); Research Diagnostics,Inc. (RDI, Flanders, N.J.); Boehringer Ingelheim Bioproducts(Heidelberg, Germany ); and BioSource (Camarillo, Calif.). Isotypic(mouse IgG-1 kappa) controls typically gave signals of ≦5% of the basalsignal. Other primary antibodies to human PECAM-1 suitable for FACS®analysis in combination with a labelled secondary antibody are availablefrom, e.g., RDI; Boehringer Ingelheim Bioproducts; Endogen (formerly TCell Diagnostics, Woburn, Mass.); PharMingen (San Diego, Calif.); OSIPharmaceuticals (formerly Oncogene Science, Uniondale, N.Y.); Serotec(Oxford, England); and Santa Cruz Biotechnology (Santa Cruz, Calif.).

The results (Table 8) indicate that, at least for ISIS 8994 and 8995,inhibition of PECAM-1 mRNA expression occurs at a fairly constant level(from about 60 to 90% inhibition, where "about" indicates ±5%) from t=4h to t=48 h. In contrast, inhibition of PECAM-1 protein expression "lagsbehind" the antisense modulation of mRNA levels; that is, a reduction inPECAM-1 protein levels is not seen until the t=24 h timepoint. Moreover,at least in the case of ISIS 8995, the antisense modulation of PECAM-1protein expression may continue to increase beyond the last timepoint ofthe experiment (t=48 h).

F. Dose Response: The dose response of PECAM-1 protein expression inHUVEC cells following 24 hours of treatment with various concentrationsof ISIS 8994 or 8996 is shown in Table 9. Levels of PECAM-1 protein weredetermined by FACS® as above. Under these conditions, both of theseantisense oligonucleotides caused about 30% inhibition of PECAM-1protein levels when applied at a concentration of 10 nM, where "about"indicates ±5%. For ISIS 8994, the degree of inhibition increased toabout 50% at 25 nM and to about 60% at concentrations of 50 to 100 nM.For ISIS 8995, the degree of inhibition increased to about 50% at 25 nMand remained at that level at higher concentrations of theoligonucleotide.

                  TABLE 5    ______________________________________    Activities of Phosphorothioate Antisense    Oligonucleotides (ASOs) Targeted to Human PECAM-1          SEQ    ISIS  ID     GENE TARGET   % mRNA   % mRNA    No    NO     REGION        EXPRESSION                                        INHIBITION    ______________________________________    Expt. 1 (t = 4 h,  ASO! = 100 nM):    basal --     LIPOFECTIN ® only                               100%      0%    8988  2      5'-UTR        62%      38%    8989  3      5'-UTR        105%     --    8990  4      5'-UTR        91%       9%    8991  5      AUG           67%      33%    8992  6      ORF           60%      40%    8993  7      ORF           66%      34%    8994  8      stop          39%      61%    8995  9      3'-UTR        53%      47%    8996  10     3'-UTR        42%      58%    Expt. 2 (t = 6 h,  ASO! = 100 nM):    basal --     LIPOFECTIN ® only                               100%      0%    11591 11     3'-UTR        50%      50%    11593 12     3'-UTR        44%      56%    11594 13     ORF           14%      86%    11595 14     3'-UTR         8%      92%    ______________________________________

                  TABLE 6    ______________________________________    Sequence Specificity of Phosphorothioate    Oligonucleotides Targeted to Human PECAM-1    ISIS  SEQ ID  GENE TARGET % mRNA    % mRNA    No    NO      REGION      EXPRESSION.sup.1                                        INHIBITION.sup.1    ______________________________________    basal --      LIPOFECTIN ®                              100%       0%                  only     8994  8      stop codon  10%       90%                  region    11914 15      sense       86%       14%                  control    11915 16      scrambled   74%       26%                  control    ______________________________________     .sup.1 Oligonucleotide concentration = 50 nM, t = 4 h.

                  TABLE 7    ______________________________________    Time Course of Response of HUVEC Cells to    PECAM-1 Antisense Oligonucleotides (ASOs)          SEQ          ID     ASO Gene           % mRNA  % mRNA    ISIS #          NO     Target Region Time Expression                                            Inhibition    ______________________________________    basal --     LIPOFECTIN ® only                               4 h  100.0%  0.0%    8994   8     stop codon    4 h  14.4%   85.6%    8994   8     "             6 h  2.4%    97.6%    8994   8     "             8 h  3.5%    96.5%    8994   8     "             12 h 5.0%    95.0%    8996  10     3'-UTR        4 h  13.6%   86.4%    8996  10     "             6 h  2.5%    97.5%    8996  10     "             8 h  4.3%    95.7%    8996  10     "             12 h 6.2%    93.8%    ______________________________________     .sup.1 Oligonucleotide concentration = 100 nM.

                  TABLE 8    ______________________________________    Time Courses of Modulation of mRNA and Protein Levels    by PECAM-1 Antisense Oligonucleotides (ASOs)          SEQ ID  ASO Gene         % mRNA  % Protein    ISIS #          NO      Target Region                              Time Expression                                           Expression    ______________________________________    basal --      LIPOFECTIN ®                               4 h 100%    100%                  only    8994  8       stop region  4 h 12%     97%    8995  9       3'-UTR       4 h 22%     92%    basal --      LIPOFECTIN ®                              24 h 100%    100%                  only    8994  8       stop region 24 h 39%     51%    8995  9       3'-UTR      24 h 30%     43%    basal --      LIPOFECTIN ®                              48 h 100%    100%                  only    8994  8       stop region 48 h 23%     55%    8995  9       3'-UTR      48 h 12%     28%    ______________________________________     .sup.1 Oligonucleotide concentration = 50 nM.

                  TABLE 9    ______________________________________    Dose Response of HUVEC Cells to PECAM-1    Antisense Oligonucleotides (ASOs)          SEQ          ID     ASO Gene    ASO    % Protein                                            % Protein    ISIS #          NO     Target      Dose   Expression                                            Inhibition    ______________________________________    basal --     LIPOFECTIN ®                              0 nM  100%     0%                 only    8994  8      stop codon  10 nM  62%     38%    8994  8      "           25 nM  48%     52%    8994  8      "           50 nM  41%     59%    8994  8      "           75 nM  40%     60%    8994  8      "           100 nM 40%     60%    8995  9      3'-UTR      10 nM  61%     39%    8995  9      "           25 nM  51%     49%    8995  9      "           50 nM  49%     51%    8995  9      "           75 nM  51%     49%    8995  9      "           100 nM 51%     49%    ______________________________________     .sup.1 t = 24 h.

Example 5

Antisense-Mediated Inhibition of Human PECAM-1 Expression by SecondGeneration Oligonucleotides

A. Design of "Second Generation" Human PECAM-1 oligonucleotides: Resultsfrom the PECAM-1-specific phosphorothioate oligonucleotides indicatethat the most active phosphorothioate oligonucleotides for modulatingPECAM-1 expression include ISIS 8994 and 8995 (SEQ ID NOs: 8 and 9,respectively). "Second generation" oligonucleotides based on theseoligonucleotides but having additional chemical modifications weredesigned and synthesized to confirm and extend these findings. Asdetailed in Table 2, ISIS 12466, 12467 and 17555 to 17560 are "gapmers"corresponding to ISIS 8994. That is, all of these oligonucleotidescomprise SEQ ID NO: 8 and have 2'-methoxyethoxy flanks or "wings" and acentral 2'-deoxy "gap" designed to support RNase H activity on thetarget mRNA molecule. ISIS 17566 and 17651 are hemimers (or "wingmers")corresponding to ISIS 8994 and have a 2'-methoxyethoxy, RNaseH-refractory 5' or 3' first portion and a 3' or 5', respectively, secondportion (the "wing") that comprises 2'-deoxy residues. Like the central"gap" portion of the gapmers, the second, 2'-deoxy portion of thehemimer oligonucleotides is designed to support RNase H activity on thetarget PECAM-1 mRNA. The 2'-methoxyethoxy portions ("wings") of theseoligonucleotides also contain 5-methyl-cytidine (m5C) residues, whichare known to increase duplex stability in some nucleic acids (see, forexample, Hoheisel et al., J. Biol. Chem., 1990, 265, 16656).

Other "second generation" compounds have sequences that are not based onany of the phosphorothioate oligonucleotides of the previous example.These include ISIS 17561-17570 (SEQ ID NOs: 19-26, respectively) and twocontrols for ISIS 17561 (ISIS 17966 and 17970, SEQ ID NOs: 28 and 29,respectively).

B. Activities of Second Generation PECAM-1 Oligonucleotides: Thechemically modified derivatives of ISIS 8994 and 8995 (SEQ ID NOs: 8 and9, respectively) were tested in the PECAM-1 protein assay (FACS®)described above with the following modifications: oligonucleotides wereadded to cells to a final concentrations of 50 nM or 100 nM, and sampleswere taken for analysis at 24 h and/or 48 h, as indicated below. 25 Theresults are presented in Table 10 (Experiments 1, 2a, 2b and 4). (InExperiment 4, ISIS 14391 was used as a control; this oligonucleotidecomprises the sequence 5'-GACGCATCGCGCCTACATCG (SEQ ID NO: 27), whereemboldened residues indicate 2'-methoxyethoxy-modified residues andunderlined "C" residues indicate 5-methylcytosine, and has aphosphorothioate backbone). Because of their ability to modulate PECAM-1expression for extended periods of time, as reflected by levels ofinhibition from ≧about 45% to 100% of PECAM-1 levels at t=24 h, and from≧about 30% to 100% at t=48h, where "about" indicates ±5%, the chemicallymodified antisense oligonucleotides targeted to human PECAM-1 arepreferred embodiments of the invention. Particularly preferred are ISIS12467, 17555, 17556, 17557, 17559, 17560, 17561 and 17567.

The additional oligonucleotides targeting human PECAM-1 were evaluatedusing the PECAM-1 protein assay. The results (Table 10, Experiment 3)indicate that all of the chemically modified oligonucleotides inhibitPECAM-1 protein levels from ≧about 45% to about 100% of PECAM-1 levelsat t=24 h, where "about" indicates ±5%. Accordingly, such chemicallymodified antisense compounds are preferred embodiments of the invention.Particularly preferred are ISIS 17561, 17562, 17563, 17564, 17565,17568, 17569 and 17570 (SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25 and 26,respectively).

The specificity of modulation of PECAM-1 by the chemically modifiedantisense oligonucleotides was confirmed using ISIS 17561 and a set ofcontrol oligonucleotides having sequences that are either mismatched orscrambled versions of that of the active oligonucleotide. Specifically,ISIS 17966 (SEQ ID NO: 28) is a control oligonucleotide having 3 basemismatches with the target PECAM-1 sequence, and ISIS 17970 (SEQ ID NO:29) is a "scrambled" control, for ISIS 17561. HUVEC cells were treatedwith these oligonucleotides at 25 nM and evaluated by FACS® as above.The results (Table 10, Experiment 5) demonstrate that neither of thecontrol oligonucleotides was capable of inhibiting PECAM-1 expressionand thus are not within the range of activity of the preferredembodiments of the invention (i.e., inhibition of PECAM-1 protein levelsfrom ≧about 35% to about 100%). In sum, the PECAM-1-modulatingactivities of the chemically modified ("second generation") antisensecompounds are sequence-specific.

                  TABLE 10    ______________________________________    Activity of "Second Generation" Antisense    Oligonucleotides Targeted to Human PECAM-1    ISIS   SEQ ID  ASO         % PROTEIN                                        % PROTEIN    No     NO      DESCRIPTION EXPRESSION                                        INHIBITION    ______________________________________    EXPT. 1 (50 nM, t = 24 h):    basal  --      LIPOFECTIN ®                               100%      0%                   only     8994  8       active-stop 81%      19%                   codon    11915  16      scrambled   125%     --                   control-8994    12466  8       8994-derived                               87%      13%                   gapmer    12467  8       8994-derived                               56%      44%                   gapmer    EXPT. 2a (100 nM, t = 24 h):    basal  --      LIPOFECTIN ®                               100%      0%                   only     8994  8       active-stop 55%      45%                   codon    11915  16      scrambled   93%       7%                   control-8994    17555  8       8994-derived                               52%      48%                   gapmer    17556  8       8994-derived                               42%      58%                   gapmer    17557  8       8994-derived                               49%      51%                   gapmer    17559  8       8994-derived                               55%      45%                   gapmer    17560  8       8994-derived                               57%      43%                   gapmer    17651  8       8994-derived                               51%      49%                   hemimer    EXPT. 2b (100 nM, t = 48 h):    basal  --      LIPOFECTIN ®                               100%      0%                   only     8994  8       active-stop 73%      27%                   codon    11915  16      scrambled   112%     --                   control-8994    17555  8       8994-derived                               73%      27%                   gapmer    17556  8       8994-derived                               64%      36%                   gapmer    17557  8       8994-derived                               67%      34%                   gapmer    17559  8       8994-derived                               73%      27%                   gapmer    17560  8       8994-derived                               73%      27%                   gapmer    17651  8       8994-derived                               70%      30%                   hemimer    EXPT. 3 (100 nM, t = 24 h):    basal  --      LIPOFECTIN ®                               100%      0%    17561  19      3'-UTR hemimer                               30%      70%    17562  20      3'-UTR hemimer                               35%      65%    17563  21      3'-UTR hemimer                               44%      56%    17564  22      stop hemimer                               37%      63%    17565  23      ORF hemimer 39%      61%    17566  8       8994-hemimer                               51%      49%    17567  9       8995-hemimer                               35%      65%    17568  24      ORF hemimer 37%      63%    17569  25      ORF hemimer 37%      63%    17570  26      ORF hemimer 36%      64%    EXPT. 4 (100 nM, t = 48 h):    basal  --      LIPOFECTIN ®                               100%     --                   only    17561  19      3'-UTR      11%      89%                   hemimer    17567  9       8995-derived                               48%      52%                   hemimer    14391  27      scrambled   84%      16%                   ICAM-1 hemimer    EXPT. 5 (25 nM, t = 48 h):    basal  --      LIPOFECTIN ®                               100%     --                   only    17561  19      3'-UTR      20%      80%                   hemimer    17966  28      17561 control                               120%     --                   (mismatch)    17970  29      17561 control                               99%       1%                   (scrambled)    ______________________________________

C. Preferred Regions for Antisense Modulation of Nucleic Acids EncodingHuman PECAM-1: The antisense compounds of the invention may be designedto be specifically hybridizable (targeted) to any portion of a nucleicacid encoding PECAM-1. However, the above results indicate that severalregions, and sequences within such regions, of nucleic acids encodinghuman PECAM-1 are particularly preferred for antisense modulation of,and are thus preferred target regions for further antisense compoundsdesigned to modulate, human PECAM-1.

1. The 5'-UTR of human PECAM-1 was the target region for ISIS 8988 (SEQID NO: 2), a preferred embodiment of the invention.

2. The AUG (start) codon region of human PECAM-1 was the target regionfor ISIS 8991 (SEQ ID NO: 5), a preferred embodiment of the invention.

3. The ORF (open reading frame) region of human PECAM-1 is the targetregion for ISIS 8992, 8993, 11594, 17565, 17569 and 17570 (SEQ ID NOs:6, 7, 13, 23, 24, 25 and 26, respectively), all of which are preferredembodiments of the invention.

4. The stop codon region of human PECAM-1 is a target region forpreferred embodiments of the invention. As shown in FIG. 2, the sequenceof this region of human PECAM-1 (bases 2305-2394 of SEQ ID NO: 1;GenBank Accession No. M28526) has a sequence (SEQ ID NO: 54)encompassing ISIS 17561, 11595, 8994, 17564, 11594 and 17568. Takentogether, results from these oligonucleotides indicate that the stopcodon region of nucleic acids that the human PECAM-1 is particularlysusceptible to antisense modulation. More specifically, antisensecompounds that specifically hybridize to SEQ ID NOs: 55 or 57 (see FIG.2) are preferred among those specifically hybridizing to the stop codonregion of a nucleic acid encoding human PECAM-1.

Particularly preferred are those antisense compounds comprising eitherof the consensus sequences derivable from the shared portions of 2 ormore antisense compounds. For example, consensus sequences sharedbetween two antisense compounds include SEQ ID NO: 58 (common to ISIS17561 and 11595), SEQ ID NO: 59 (ISIS 11595 and 8994) and SEQ ID NO: 60(ISIS 8994 and 17564). Especially preferred are antisense compoundscomprising consensus sequences derivable from the shared portions of 3or more antisense compounds, i.e., 5'-GTGCATC (common to ISIS 17561,11595 and 8994) and 5'-TGGCC (common to ISIS 11595, 8994 and 17564).

5. The 3'-UTR (untranslated region) of human PECAM-1 is a target regionfor preferred embodiments of the invention. As shown in FIG. 3, thesequence of this region of human PECAM-1 (bases 2436-2520 of SEQ ID NO:1; GenBank Accession No. M28526) has a sequence (SEQ ID NO: 61)encompassing ISIS 8996, 11593, 17563, 8995 and 11591. Taken together,results from these oligonucleotides indicate that the 3'-untranslatedregion (3'-UTR) of nucleic acids that the human PECAM-1 is particularlysusceptible to antisense modulation. More specifically, antisensecompounds that specifically hybridize to SEQ ID NOs: 62, 64 or 66 (seeFIG. 3) are preferred among those specifically hybridizing to the 3'-UTRof a nucleic acid encoding human PECAM-1.

Particularly preferred are those antisense compounds comprising eitherof the consensus sequences derivable from the shared portions of 2 ormore antisense compounds. For example, a consensus sequence sharedbetween two antisense compounds is SEQ ID NO: 65 (common to ISIS 8996and 11593). Especially preferred are antisense compounds comprisingconsensus sequences derivable from the shared portions of 3 or moreantisense compounds, i.e., SEQ ID NO: 67 (common to ISIS 17563, 8995 and11591).

Example 6

Antisense-Mediated Inhibition of Murine PECAM-1 Expression byPhosphorothioate Oligonucleotides

A. Murine PECAM-1-Specific Probes: In initial screenings of a set ofoligonucleotides derived from the murine PECAM-1 sequence (Table 3) forbiological activity, a radiolabelled mouse PECAM-1-specific PCR productwas prepared using RNA from bEND.3 cells and a pair of phosphodiesteroligonucleotides as PCR primers, i.e.,

5'-GAGGACACTTCCACTTCTGTGT (ISIS 14244, SEQ ID NO: 51); and

5'-GGCCTGGTACCGATCCAGGTGT (ISIS 14245, SEQ ID NO: 52),

in PCR reactions using a PRIME-A-GENE labeling kit (Promega, Madison,Wis.). The resultant ³² P-cytidine-radiolabelled 898-bp producthybridizes to the open reading frame of murine PECAM-1 (nucleotides1257-2155 of GenBank accession No. L06039, locus name "MUSPECAM1").Alternatively, however, other PCR probes can be prepared, or one or moreof the oligonucleotides of Tables 3 or 4 can be detectably labeled andused as a murine PECAM-1-specific probe, using methods known in the art.

B. Activities of Murine PECAM-1 Oligonucleotides: In an initial screenfor active compounds capable of modulating PECAM-1 expression, bEND.3cells were treated with 200 nM of a series of PECAM-1-specificphosphorothioate oligonucleotides (ISIS 13983-13999, SEQ ID NOs: 31-47)and PECAM-1 mRNA levels were determined by Northern analysis. The datafrom the screening (Table 11) indicate the following results.Oligonucleotides showing activity in this assay, as reflected by levelsof inhibition from ≧about 35% to about 100% of PECAM-1 mRNA levels,where "about" indicates ±5%, include ISIS 13985, 13986, 13988, 13989,13990, 13991, 13992, 13993, 13994, 13995 and 13996 (SEQ ID NOs: 33, 34,36, 37, 38, 39, 40, 41, 42, 43 and 44, respectively). Theseoligonucleotides are thus preferred embodiments of the invention formodulating murine PECAM-1 expression. Oligonucleotides showing levels ofinhibition of from ≧about 50% to about 100% of PECAM-1 mRNAs in thisassay, where "about" indicates ±5%, include ISIS 13989, 13990, 13991,13992, 13993, 13994, 13995 and 13996 (SEQ ID NOs: 37, 38, 39, 40, 41,42, 43 and 44, respectively). These oligonucleotides are thus morepreferred embodiments of the invention for modulating murine PECAM-1expression. Among the phosphorothioate oligonucleotides initiallyscreened, the most preferred embodiments of the invention are thoseshowing levels of inhibition of from ≧about 65% to about 100% of PECAM-1mRNAs in this assay, where "about" indicates ±5%, i.e., ISIS 13990,13991, 13992 and 13995 (SEQ ID NOs: 38, 39, 40 and 43, respectively).

C. Sequence Specificity: The specificity of modulation of PECAM-1 by theantisense oligonucleotides was confirmed using ISIS 13989 and a controloligonucleotide having a scrambled version of the sequence of the activeoligonucleotide. Specifically, ISIS 14449 (SEQ ID NO: 50) is a"scrambled" control for ISIS 8994. BEND.3 cells were treated with theseoligonucleotides at 50 nM and evaluated by FACS analysis at t=24 h. Inthese experiments, murine PECAM-1 protein levels were measured by FACSOanalysis using a monoclonal antibody to murine PECAM-1 conjugated toFITC (Pharmingen, San Diego, Calif.). Isotypic (rat IgG-2a kappa)FITC-labelled controls typically gave signals of ≦5% of the basalsignal. Other primary antibodies to murine PECAM-1 suitable for FACSanalysis in combination with a labelled secondary antibody are availablefrom, e.g., Santa Cruz Biotechnology (Santa Cruz, Calif.); Endogen(formerly T Cell Diagnostics, Woburn, Mass.); PharMingen (San Diego,Calif.); and Paesel & Lorei (Hanau, Germany).

The results (Table 12) demonstrate that the control (scrambled)oligonucleotide was not capable of modulating PECAM-1 expression (5%inhibition) whereas the active compound, ISIS 13989, caused about 50%inhibition. Thus, the PECAM-1-modulating activity of the activeantisense compounds is sequence-specific.

D. Time Course: The time course of inhibition of PECAM-1 mRNA expressionin bEND.3 cells following treatment with 300 nM of ISIS 13989, 13991 and13995, as measure by FACS® analysis, is shown in Table 13. Twenty-fourhours after treatment with ISIS 13989, the level of inhibition of murinePECAM-1 was about 39% (t=24 h) , rose to about 70% inhibition at t=48 h,and subsequently decreased to about 15% at t=72 h, where "about"indicates ±5%. A similar temporal pattern of modulation was seen forISIS 13991 and 13995.

E. Dose Response: The dose response of PECAM-1 protein expression inbEND.3 cells following treatment with various concentrations of ISIS13989, 13991 or 13995 is shown in Table 14. Levels of PECAM-1 proteinwere determined by FACS . Under these conditions and at a concentrationof 37.5 nM, ISIS 13989 caused about 40% inhibition, whereas ISIS 13991and 13995 caused about 20% inhibition, of PECAM-1 protein levels (where"about" indicates ±5%). For ISIS 13989, the degree of inhibitionincreased to about 50% at 75 nM, to about 60% at 150 nM and to about 70%at 300 nM. For ISIS 13991, the degree of inhibition increased to about25% at 75 nM, to about 45% at 150 nM and to about 60% at 300 nM. ForISIS 13995, the degree of inhibition increased to about 30% at 75 nM andto about 60% at 150 nM and 300 nM.

                  TABLE 11    ______________________________________    Activities of Phosphorothioate Oligonucleotides    Targeted to Murine PECAM-1          SEQ    ISIS  ID     GENE TARGET   % mRNA   % mRNA    No    NO     REGION        EXPRESSION                                        INHIBITION    ______________________________________    basal --     LIPOFECTIN ® only                               100%      0%    13983 31     ORF           82%      18%    13984 32     ORF           75%      25%    13985 33     5'-UTR        66%      34%    13986 34     5'-UTR        66%      34%    13987 35     AUG           125%     --    13988 36     ORF           64%      36%    13989 37     ORF           50%      50%    13990 38     ORF           37%      63%    13991 39     ORF           25%      75%    13992 40     ORF           37%      63%    13993 41     ORF           46%      54%    13994 42     ORF           45%      55%    13995 43     stop          34%      66%    13996 44     3'-UTR        47%      53%    13997 45     3'-UTR        88%      12%    13998 46     3'-UTR        92%       8%    13999 47     3'-UTR        107%     --    ______________________________________     .sup.1 Oligonucleotide concentration = 200 nM, t = 4 h.

                  TABLE 12    ______________________________________    Sequence Specificity of Phosphorothioate Antisense    Oligonucleotides (ASOs) Targeted to Murine PECAM-1          SEQ    ISIS  ID     ASO GENE      % PROTEIN                                        % PROTEIN    No    NO     TARGET REGION EXPRESSION                                        INHIBITION    ______________________________________    basal --     LIPOFECTIN ® only                               100%     0%    13989 37     stop codon region                               49%      51%    14449 50     13989-scrambled                               95%      5%                 control    ______________________________________     .sup.1 bEND.3 cells treated with 50 nM (final concentration) of indicated     oligonucleotides; samples taken at t = 24 h.

                  TABLE 13    ______________________________________    Time Course of Response of bEND.3 Cells to    Murine PECAM-1 Antisense Oligonucleotides (ASOs)          SEQ          ID     ASO Gene Target    % Protein                                            % Protein    ISIS #          NO     Region        Time Expression                                            Inhibition    ______________________________________    basal --     LIPOFECTIN ® only                               24 h 100.0%  0.0%    basal --     "             48 h 100.0%  0.0%    basal.sup.2          --     "             72 h 100.0%  0.0%    13989 37     ORF           24 h 60.8%   39.2%    13989 37     "             48 h 31.2%   68.8%    13989 37     "             72 h 85.1%   14.9%    13991 39     ORF           24 h 64.7%   35.3%    13991 39     "             48 h 34.2%   65.8%    13991 39     "             72 h 97.3%   2.7%    13995 43     stop codon    24 h 52.7%   47.3%    13995 43     "             48 h 28.4%   71.6%    13995 43     "             72 h 83.0%   17.0%    ______________________________________     .sup.1 bEND.3 cells treated with 300 nM (final concentration) of indicate     oligonucleotides.     .sup.2 Derived from two samples; all other data points are averages     (means) of determinations from three samples.

                  TABLE 14    ______________________________________    Dose Response of Murine bEND.3 Cells to    Murine PECAM-1 Antisense Oligonucleotides (ASOs)          SEQ          ID     ASO Gene           % Protein                                            % Protein    ISIS #          NO     Target      Dose   Expression                                            Inhibition    ______________________________________    basal --     LIPOFECTIN ®                               0 nM  100%     0%                 only    13989 37     ORF         37.5 nM                                    59.6%   40.4%    13989 37     "             75 nM                                    46.9%   53.1%    13989 37     "            150 nM                                    39.5%   60.5%    13989 37     "            300 nM                                    34.7%   66.3%    13991 39     ORF         37.5 nM                                    80.5%   19.5%    13991 39     "             75 nM                                    76.5%   23.5%    13991 39     "            150 nM                                    55.0%   45.0%    13991 39     "            300 nM                                    38.5%   61.5%    13995 43     stop codon  37.5 nM                                    80.0%   20.0%    13995 43     "             75 nM                                    68.8%   31.2%    13995 43     "            150 nM                                    39.2%   60.8%    13995 43     "            300 nM                                    44.0%   56.0%    ______________________________________     .sup.1 t = 48 h.

Example 7

Antisense-Mediated Inhibition of Murine PECAM-1 Expression by SecondGeneration Oligonucleotides

A. Design of "Second Generation" Human PECAM-1 oligonucleotides: Resultsfrom the murine PECAM-1-specific phosphorothioate oligonucleotidesindicate that one of the preferred phosphorothioate oligonucleotides formodulating PECAM-1 expression is ISIS 13989 (SEQ ID NO: 37). "Secondgeneration" oligonucleotides having the same nucleobase sequence as ISIS13989 but having additional chemical modifications were designed andsynthesized to confirm and extend these findings. As detailed in Table4, ISIS 17050, 17051, 17052, 17053 and 20380 are "gapmers" having thesame nucleobase sequence as ISIS 13989; all of these oligonucleotideshave 2'-methoxyethoxy flanks or "wings" and a central 2'-deoxy "gap"designed to support RNase H activity on the target mRNA molecule. ISIS17049 and 17054 are hemimers having the same nucleobase sequence as ISIS13989 and having a 2'-methoxyethoxy, RNase H-refractory 3' or 5' firstportion and a 5' or 3', respectively, second portion (the "wing") thatcomprises 2'-deoxy residues. Like the central "gap" portion of thegapmers, the second, 2'-deoxy portion of the hemimer oligonucleotides isdesigned to support RNase H activity on the target PECAM-1 mRNA. The2'-methoxyethoxy portions ("wings") of these oligonucleotides alsocontain 5-methyl-cytidine (m5C) residues.

B. Activities of Second Generation Murine PECAM-1 Oligonucleotides: Thechemically modified derivatives of ISIS 13989 (SEQ ID NO: 37) weretested in the PECAM-1 protein assay (FACS®) described above with thefollowing modifications: oligonucleotides were added to cells to a finalconcentrations of 50 nM, and samples were taken for analysis at 24 h.The results are presented in Table 15. Because of their ability tomodulate PECAM-1 expression to the same or greater degree than theparent phosphorothioate antisense compound ISIS 13989 (as reflected bylevels of inhibition from ≧about 50% to 100% of PECAM-1 levels at t=24h, where "about" indicates ±5%), the chemically modified antisenseoligonucleotides targeted to murine PECAM-1 are preferred embodiments ofthe invention. Preferred are ISIS 17049, 17050, 17051, 17052, 17053 and17054. Particularly preferred are those antisense compounds causinglevels of inhibition from ≧about 70% to 100% of PECAM-1 levels at t=24h, i.e., ISIS 17050, 17051, 17052 and 17053.

                  TABLE 15    ______________________________________    Activity of "Second Generation" Antisense    Oligonucleotides Targeted to Murine PECAM-1          SEQ    ISIS  ID     GENE          % PROTEIN                                        % PROTEIN    No    NO     TARGET REGION EXPRESSION                                        INHIBITION    ______________________________________    basal --     LIPOFECTIN ® only                               100%      0%    13989 37     stop codon region                               49%      51%                 (phosphorothioate)    17049 37     13989-derived 44%      56%                 3'-hemimer    17050 37     13989-derived 25%      75%                 gapmer    17051 37     13989-derived 28%      72%                 gapmer    17052 37     13989-derived 32%      68%                 gapmer    17053 37     13989-derived 33%      67%                 gapmer    17054 37     13989-derived 40%      60%                 5'-hemimer    ______________________________________     .sup.1 bEND.3 cells treated with 50 nM (final concentration) of indicated     oligonucleotides; samples taken at t = 24 h.

Example 8

Animal Model for Antisense Modulation of PECAM-1

The present invention provides compositions and methods for modulatingthe transmigration of leukocytes and thus controlling relatedundesirable immune responses such as, e.g., inflammation. In order totest the ability of the antisense compounds of the invention to modulatecellular transmigration, a variation of the thioglycollate-inducedperitonitis model of Watson et al. (Nature, 1991, 349, 164; see alsoLiao et al., J. Exp. Med., 1997, 185, 1349, and Bogen et al., J. Exp.Med., 1994, 179, 1059) was used in which lipopolysaccharide (LPS) issubstituted for thioglycollate. Groups (n=5 per group) of female C57Black 6 mice were given saline or various doses (0.3, 1, 3, 10 or 30mg/kg) of antisense oligonucleotide by i.v. daily for seven days andthen challenged with 25 ug of LPS from Salmonella typhosa (DIFCOlaboratories, Livonia, Mich.) per mouse given i.p. Peritoneal lavageswere performed 18 hours later and cells were prepared using CYTOSPIN®cytocentrifuges (Shandon Lipshaw, Pittsburgh, Pa.). To determine theproportion of polymorphonuclear neutrophil granulocytes (PMNs) presentin the peritoneal fluid, the cells were stained with Wright/Giemsa stain(Sigma Chemical Co., St. Louis, Mo.) and different cell types, includingPMNs, were counted.

The results (Table 16) indicate that treatment with ISIS 13989 (SEQ IDNO: 37) reduced the transmigration of PMNs into the peritoneal fluid.Specifically, at a dose of 3 mg/kg, the absolute percentage of PMNsdecreased to about 70%, where "about" indicates ±5%. At doses of 10 and30 mg/kg, animals treated with ISIS 13989 had peritoneal PMN percentagesof about 40% and 25%, respectively. In contrast, animals treated withISIS 14449, a scrambled control for ISIS 13989, showed PMN levels ofabout 65% and 55% at doses of 3 and 30 mg/kg, respectively. Theseresults demonstrate the ability of the antisense compounds of theinvention to modulate the transmigration of leukocytes and relatedundesired immune responses.

                  TABLE 16    ______________________________________    Activity of PECAM-1-Modulating Antisense    Compounds in Animal Model of Peritonitis                          Number of PMNs                                       Relative    ISIS No.            Dosing Schedule                          in Peritoneum                                       Response    ______________________________________    (saline)            i.v. daily × 7 days                          1.3 × 10.sup.6                                       1.00    13989   0.3 mg/kg (as above)                          1.2 × 10.sup.6                                       0.92    13989   1.0 mg/kg (as above)                          1.3 × 10.sup.6                                       1.00    13989   3.0 mg/kg (as above)                          9.0 × 10.sup.5                                       0.69    13989   10 mg/kg (as above)                          5.1 × 10.sup.5                                       0.39    13989   30 mg/kg (as above)                          3.4 × 10.sup.5                                       0.26    14449   3.0 mg/kg (as above)                          8.3 × 10.sup.5                                       0.63    14449   30 mg/kg (as above)                          7.1 × 10.sup.5                                       0.54    ______________________________________

Example 9

Therapeutic Methods

The antisense compounds of the invention result in immunosuppressive andanti-inflammatory effects in vivo and may be used by those skilled inthe art to provide prophylactic, palliative and therapeutic benefit toan animal, including a human, in need of such effects. The antisensecompounds of the invention are also evaluated for their ability toinhibit the metastasis of cancer cells and are used to provideprophylactic, palliative or therapeutic relief from hyperproliferativedisorders. Therapeutic methods using the antisense compounds of theinvention include, but are not limited to, the following examples.

A. Modulation of Undesirable Immunoresponsive Events:

The present invention provides a method of modulating immunoresponsiveevents that are mediated or influenced, either directly or indirectly,by PECAM-1 in an animal. Such immunoresponsive events can lead toundesirable effects such as, e.g., inflammation. The method ofmodulating immunoresponsive events mediated or influenced by PECAM-1comprises administering one or more of the antisense compounds of theinvention (or a combination thereof with one or more anti-inflammatoryor immunosuppressive non-antisense-based agents or NABAs; see below), ina pharmaceutical preparation if required, to the animal. Some specifictherapeutic modalities for the antisense compounds of the inventionfollow as examples but are not intended to limit the scope of theinvention.

1. Diapedesis: The present invention provides a method of modulatingPECAM-1-mediated diapedesis in an animal comprising administering one ormore of the antisense compounds of the invention, or a combinationthereof with one or more anti-inflammatory or immunosuppressive agents,in a pharmaceutical preparation if required, to the animal. Diapedesis,the transendothelial migration of immunoresponsive cells (such asleukocytes) from the circulatory system into injured or infectedtissues, is thought to be a key event in inflammatory injury (for areview, see Albelda et al., FASEE J., 1994, 8, 504). Several lines ofevidence suggest that PECAM-1 is particularly important for thediapedesis of some cells such as leukocytes (white blood cells), whichinclude, for example, polymorphonuclear neutrophil granulocytes (PMNs orneutrophils) and some macrophages such as monocytes, and lymphocytessuch as natural killer (NK) cells and T lymphocytes (T cells). First,antibodies to PECAM-1 block the transmigration of neutrophils(Vaporciyan et al., Science, 1993, 262, 1580), monocytes (Muller et al.,J. Exp. Med., 1993, 178, 449; Shen et al., Am. J. Physiol., 1996, 270,H1624), leukocytes (Muller, Agents Actions Suppl., 1995, 46, 147) and NKcells (Berman et al., J. Immunol., 1996, 1516, 1515). Second, apolypeptide corresponding to a portion of PECAM-1 blockstransendothelial migration of PMNs and monocytes both in vitro and invivo (Liao et al., J. Exp. Med., 1997, 185, 1349). Administration of theantisense compounds of the invention, as part of an appropriatepharmaceutical composition if required, to an animal is expected toinhibit diapedesis and subsequent undesired immunoresponsive events suchas, for example, inflammation and inflammatory damage. Such treatmentmay be in combination with one or more anti-inflammatory and/orimmunosuppressive NABAs and, additionally or alternatively, with one ormore additional antisense compounds targeted to, for example, a CAMprotein, a B7 protein, or a protein kinase C (see below). Suchadministration can be systemic or directly to the site(s) of diapedesis,inflammation and/or inflammatory damage. The antisense compounds of theinvention are evaluated for their ability to modulate diapedesis andsubsequent undesired inflammation and/or inflammatory damage using, forexample, the assays described in the references cited in this section,the in vitro flow model of Luscinskas et al. (J. Immuncol., 1996, 157,326), the mouse model using carrageenan-soaked sponges described inExample 22 of U.S. Pat. No. 5,514,788 to Bennett et al. (herebyincorporated by reference) and/or other relevant animal models.

2. Acute Inflammation: The present invention also provides a method ofmodulating acute inflammation, such as occurs in peritonitis, in ananimal comprising administering one or more of the antisense compoundsof the invention, or a combination thereof with one or moreanti-inflammatory or immunosuppressive agents, in a pharmaceuticalpreparation if required, to the animal. A murine monoclonal antibody toPECAM-1 blocks acute inflammation in an in vivo (murine) model ofperitonitis (Bogen et al., J. Exp. Med., 1994, 179, 1059) and,similarly, a rabbit polyclonal antibody inhibits leukocytetransmigration in glycogen-induced peritonitis in cats (Murohara et al.,J. Immunol., 1996, 156, 3550). Administration of the antisense compoundsof the invention, as part of an appropriate pharmaceutical compositionif required, to an animal is expected to inhibit undesired acuteinflammation and resulting inflammatory damage. Such administration canbe systemic or directly to the site(s) of inflammation. Such treatmentmay be in combination with one or more anti-inflammatory and/orimmunosuppressive NABAs and, additionally or alternatively, with one ormore additional antisense compounds targeted to a CAM protein, a B7protein, or a protein kinase C (see below). The antisense compounds ofthe invention are evaluated for their ability to modulate acuteinflammation and subsequent events using, for example, the assaysdescribed in the reference cited in this section or known in the artand/or appropriate animal models.

3. Corneal Inflammation: The present invention also provides a method oftreating corneal inflammation in an animal comprising administering oneor more of the antisense compounds of the invention, or a combinationthereof with one or more anti-inflammatory or immunosuppressive agents,in a pharmaceutical preparation if required, to the animal. PECAM-1 hasbeen stated to be found at zones of marked inflammation in specimensfrom a broad range of corneal inflammatory diseases (Goldberg et al.,Opthamology, 1994, 101, 161). Administration of the antisense compoundsof the invention, as part of an appropriate pharmaceutical compositionif required, to an animal is expected to inhibit undesired cornealinflammation and resulting inflammatory damage. Such administration canbe systemic or directly to the cornea(s) exhibiting inflammation. Suchtreatment may be in combination with one or more anti-inflammatoryand/or immunosuppressive NABAs and, additionally or alternatively, withone or more additional antisense compounds targeted to a CAM protein, aB7 protein, or a protein kinase C (see below). The antisense compoundsof the invention are evaluated for their ability to modulate cornealinflammation and subsequent events using one or more assays known in theart and/or one or more appropriate animal models.

4. Allograft Rejection and GVHD: The present invention also provides amethod of avoiding allograft rejection including treating or preventinggraft versus host disease (GVHD) in an animal comprising administeringone or more of the antisense compounds of the invention, or acombination thereof with one or more anti-inflammatory orimmunosuppressive agents, in a pharmaceutical preparation if required,to the animal. At least one study suggests that nucleic acids encodinghuman PECAM-1 are polymorphic, and that, when donor and recipientPECAM-1 genotypes are not matched in bone marrow transplants, the riskof acute GVHD increases (Behar et al., New England J. Med., 1996, 334,286). Accordingly, administration of one or more of thePECAM-1-modulating antisense compounds of the invention (in combinationwith other agents and as part of an appropriate pharmaceuticalcomposition if required) to an animal is expected to modulate allograftrejection and GVHD.

Such administration can be systemic or directly to the area(s) of thetransplanted tissue(s) or organ(s), or administered ex vivo to tissue(s)or organ(s) prior to their transplantation. Such treatment may be incombination with one or more anti-inflammatory/immunosuppressive NABAsand, additionally or alternatively, with one or more additionalantisense compounds targeted to a CAM protein, a B7 protein, or aprotein kinase C (see below). The antisense compounds of the inventionare evaluated for their ability to modulate allograft rejection usingone or more assays known in the art and/or one or more appropriateanimal models (see, e.g., Stepkowski et al., J. Immunol., 1994, 153,5336, and Example 21 in U.S. Pat. No. 5,514,788 to Bennett et al.,hereby incorporated by reference).

5. Arthritis: The present invention also provides a method of treatingvarious forms of arthritis in an animal comprising administering one ormore of the antisense compounds of the invention, or a combinationthereof with one or more anti-inflammatory or immunosuppressive agents,in a pharmaceutical preparation if required, to the animal. Suchadministration can be systemic or directly to involved tissues such as,e.g., synovial fluid. Increased expression of CAMs, including ELAM-1,VCAM-1, ICAM-1 and PECAM-1 has been detected in synovial fluid frompatients having rheumatoid arthritis (Tak et al., Clin. Immunol.Immunopathol., 1995, 77, 236). Such forms of arthritis include, forexample, autoimmune forms of arthritis, including some forms ofrheumatoid arthritis (RA), psoriatic arthritis (PA) and ankylosingspondylitis (AS) non-autoimmune forms of RA, PA and AS; infectiousarthritis, such as results from infection with spirochetes (Lymedisease, also known as LD or Lyme arthritis, is caused by Borreliaburgdorferi, and some instances of Reiter's syndrome, RS, appear to beassociated with Chlamydia trachomatis), bacterial infection(staphylococci such as Hemophilus influenzae, streptococci, pneumococci,gram-negative bacilli and the like), viral infection (e.g., rubella,mumps, human parvovirus or hepatitis B), and/or fungal infection (e.g.,Sporothrix schenckii, Coccidioides immitis, Blastomyces dermatididis orCandida albicans) (see The Merck Manual of Diagnosis and Therapy, 15thEd., pp. 1239-1267, Berkow et al., eds., Rahay, N.J., 1987). Suchtreatment may be in combination with one or more additional antisensecompounds or anti-inflammatory/immunosuppressive NABAs and, additionallyor alternatively, when the arthritis to be treated results at least inpart from infection of the animal by a pathogen, with one or moreantibiotics (see below). The antisense compounds of the invention areevaluated for their ability to modulate arthritis and inflammatorydamage resulting therefrom using one or more assays known in the artand/or one or more appropriate animal models (see, e.g., published PCTapplication No. WO 95/32285 to Benoist et al.).

6. Inflammatory Disorders of the Bowel: The present invention alsoprovides a method of treating various inflammatory disorders of thebowel in an animal comprising administering one or more of the antisensecompounds of the invention, or a combination thereof with one or moreanti-inflammatory or immunosuppressive agents, in a pharmaceuticalpreparation if required, to the animal. Such disorders include, forexample, Crohn's disease (CD) and other forms of regional enteritis; andvarious forms of colitis including ulcerative colitis (UC) andgranulomatous, ischemic and radiation colitis (see The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 797-806, Berkow et al., eds.,Rahay, N.J., 1987). Such treatment may be in combination with one ormore additional antisense compounds or anti-inflammatory and/orimmunosuppressive NABAs (see below). The antisense compounds of theinvention are evaluated for their ability to modulate a inflammatorydisorder of the bowel using one or more assays known in the art and/orone or more appropriate animal models (see, e.g., okayasu et al.,Gastroenterol., 1990, 98, 694, and Example 20 in U.S. Pat. No. 5,514,788to Bennett et al., hereby incorporated by reference).

7. Autoimmune Diseases and Disorders: The present invention alsoprovides a method of treating various autoimmune diseases and disordersincluding but not limited to autoimmune thyroid disorders; autoimmuneforms of arthritis; multiple sclerosis (MS); some forms of juvenilediabetes mellitus; myasthenia gravis; pemphigus vulgaris; and systemiclupus erythematosus (SLE or lupus) (for a review of autoimmunedisorders, see Steinman, Sci. Amer., 1993, 269, 107). A preferredembodiment of the invention involves the treatment or prevention ofautoimmune thyroid disorders, such as, e.g., Graves' Disease(thyrotoxicosis), Hashimoto's disease and De Quervain thyroiditis, asPECAM-1 has been observed to be expressed on cells involved in suchconditions (Marazuela et al., Clin. Exp. Immunol., 1995, 102, 328;Aubert et al., Clin. Immunol. Immunopathol., 1995, 76, 170).Administration of the antisense compounds of the invention, as part ofan appropriate pharmaceutical composition if required, to an animal isexpected to prevent or inhibit the development of the autoimmune diseaseand subsequent undesired events. Such treatment may be in combinationwith one or more anti-inflammatory/immunosuppressive NABAs and,additionally or alternatively, with one or more additional antisensecompounds targeted to, for example, a CAM protein, a B7 protein, or aprotein kinase C (see below). Such administration can be systemic ordirectly to a specific tissue, depending on the nature of the disorder.For example, systemic administration might be more appropriate for SLE,whereas direct administration to the thyroid gland or adjacent tissuesmight be more efficacious in the case of Graves' Disease. The antisensecompounds of the invention are evaluated for their ability to prevent orinhibit autoimmune diseases using appropriate assays and animal modelsknown to those skilled in the art (see, for example, Burkhardt et al.,Rheumatol. Int., 1997, 17, 91).

B. Cardiovascular Disorders: The present invention provides a method ofmodulating undesirable events that are mediated or influenced, eitherdirectly or indirectly, by PECAM-1 in an animal during or followingcardiovascular injury or during the course of a cardiovascular diseaseor disorder. The method of modulating immunoresponsive events mediatedor influenced by PECAM-1 comprises administering one or more of theantisense compounds of the invention, in a pharmaceutical preparation ifrequired, to the animal. Some specific therapeutic modalities for theantisense compounds of the invention follow as examples but are notintended to limit the scope of the invention.

1. Myocardial Ischemia/Reperfusion Injury: The present inventionprovides a method of reducing or preventing myocardialischemia/reperfusion (MI/R) injury in an animal comprising administeringone or more of the antisense compounds of the invention, or acombination thereof with one or more anti-inflammatory orimmunosuppressive agents, in a pharmaceutical preparation if required,to the animal. Coronary artery reperfusion is an effective treatment forpatients with acute myocardial infarction. However, reperfusion itselfoften leads to enhanced injury of myocardial tissue. Transmigration ofPMNs into the ischemic myocardium is believed to play some role inmyocardial ischemia/reperfusion injury (Entman et al., FASEB J., 1991,5, 2529; Lefer et al., FASEB J., 1991, 5, 2029). Antibodies to PECAM-1attenuate MI/R injury in vivo in feline and rodent models (Gumina etal., Circ., 1996, 94, 3327; Murohara et al., J. Immunol., 1996, 156,3550). Administration of the antisense compounds of the invention, aspart of an appropriate pharmaceutical composition if required, to ananimal is expected to modulate MI/R injury. Such administration can besystemic or directly to the circulatory system. Such treatment may be incombination with one or more additional antisense compounds targeted to,e.g., a CAM protein, a B7 protein, or a protein kinase C (see below).The antisense compounds of the invention are evaluated for their abilityto modulate MI/R injury using one or more assays known in the art and/orone or more appropriate animal models such as, for example, thosedescribed in the references cited in this section.

2. Stroke-Related Damage: The present invention provides method ofreducing leukocyte-induced damage in an animal during or following astroke or series of strokes, or of preventing leukocyte-induced damagefrom a stroke in an animal. known to be prone to having strokes, in ananimal comprising administering one or more of the antisense compoundsof the invention, in a pharmaceutical preparation if required, to theanimal. A stroke is a blockage or hemorrhage of a blood vessel in orleading to the brain (e.g., aneurysmal subarachnoid hemorrhage) thatcauses inadequate blood supply (ischemia) to the brain. After brainischemia, leukocytes adhere to the perturbed vascular endothelium andare believed to aggravate reperfusion injury eventually leading, in somecases, to chronic vasospasm (i.e., a sudden constriction of an artery orvein), which in turn can have serious and undesired consequences.Adhesion molecules, including ICAM-1, are upregulated in acuteinfarctions from brain sections of human subjects who died within 18days of ischemic stroke (Lindsberg et al., Circ., 1996, 94, 939). In ananimal model, a monoclonal antibody to ICAM-1 inhibited vasospasm(Oshiro et al., Stroke, 1997, 28, 2031). Administration of the antisensecompounds of the invention, as part of an appropriate pharmaceuticalcomposition if required, to an animal is expected to stroke-relatedinjuries. Such administration can be systemic or directly to thecirculatory system. Such treatment may be in combination with one ormore additional antisense compounds targeted to a CAM protein, a B7protein, or a protein kinase C (see below). Alternatively, in thismethod of the invention, compounds targeted to a CAM protein, a B7protein, or a protein kinase can be used without an antisense compoundtargeted to PECAM-1, or in combination with each other. The antisensecompounds are evaluated for their ability to modulate stroke-relatedinjury using one or more assays known in the art and/or one or moreappropriate animal models such as, for example, those described in thereferences cited in this section.

C. Treatment of Hyperproliferative Disorders: Patients having benigntumors, and primary malignant tumors that have been detected early inthe course of their development, may often be successfully treated bythe surgical removal of the benign or primary tumor. If unchecked,however, cells from malignant tumors are spread throughout a patient'sbody through the processes of invasion and metastasis. Invasion refersto the ability of cancer cells to detach from a primary site ofattachment and penetrate, e.g., an underlying basement membrane.Metastasis indicates a sequence of events wherein (1) a cancer celldetaches from its extracellular matrices, (2) the detached cancer cellmigrates to another portion of the patient's body, often via thecirculatory system, and (3) attaches to a distal and inappropriateextracellular matrix, thereby creating a focus from which a secondarytumor can arise. Normal cells do not possess the ability to invade ormetastasize and/or undergo apoptosis (programmed cell death) if suchevents occur (Ruoslahti, Sci. Amer., 1996, 275, 72).

Disseminating precancerous or cancerous cells often display ectopicexpression of adhesion molecules which may facilitate step (3) of themetastatic process as described above. Examples of such adhesionmolecules include ICAM-1, PECAM-1 and other CAMs (for a review, see Tanget al., Invasion Metastasis, 1994, 14, 109). Thus, modulation of one ormore CAMs using the antisense compounds of the invention may result in adecreased ability of disseminating cancer cells to attach to a distaland/or inappropriate matrix, thereby modulating metastasis of theprimary tumor. Moreover, because PECAM-1 apparently also helps regulatethe expression and/or activity of other molecules involved in cell-cellinteractions (Litwin et al., J. Cell Biol., 1997, 139, 219), modulationof PECAM-1 by the oligonucleotides of the present disclosure will alsoresult in the modulation of expression of molecules involved incell-cell interactions, further limiting the metastatic ability oftumors.

The present invention thus also provides a method of modulating orpreventing metastasis in an animal comprising administering one or moreof the antisense compounds of the invention, in a pharmaceuticalpreparation if required, to the animal. Such treatment may be incombination with one or more additional antisense compounds oranticancer chemotherapeutic NABAs (see below). The antisense compoundsof the invention are evaluated for their ability to modulate metastasisusing one or more assays known in the art and/or one or more appropriateanimal models (see, e.g., Examples 16-18 in U.S. Pat. No. 5,514,788 toBennett et al., hereby incorporated by reference). As another example,cell line number CRL-11448 from the American Type Culture Collection(Rockville, Md.) is an PECAM-1/CD31⁺ endothelial cell line derived froma patient with Kaposi's sarcoma that forms metastatic tumors inimmunodeficient mice. CRL-11448 can be used to evaluate the ability ofthe PECAM-1-targeted antisense compounds of the invention to modulatemetastatic events in vivo.

D. Combination Therapies and Compositions

If desired, the therapeutic antisense modulation of the expression ofPECAM-1 (or another CAM) can be combined with additional therapies inorder to achieve a requisite level of interference with, or preventionof, undesirable disorders or diseases. Such combinations can be carriedout, for example, by simultaneously administering (a) two or moreantisense compounds targeted to two or more CAMs, (b) two or moreantisense compounds, one targeted to a CAM and one another genetictarget or (c), when treating an animal having inflammation, an antisensecompound targeted to a CAM in combination with a non-antisense-basedanti-inflammatory or immunosuppressive agent. If an animal having ahyperproliferative disease or disorder is to be treated, the antisensecompound targeted to a CAM may be combined with a non-antisense-basedchemotherapeutic agent. When used with the antisense compounds of theinvention, such non-antisense-based agents (NABAs) may be used in simplecombination (e.g., administration of a NABA and an antisense compound),sequentially (e.g., administration of a first NABA and an antisensecompound for a period of time followed by administration of a secondNABA and an antisense compound), or in combination with one or moreother such non-antisense-based agents or physical treatments (e.g.,administration of a NABA and an antisense compound, or, in the treatmentof hyperproliferative disorders for example, administration of one ormore NABAs and antisense compounds in combination with radiotherapy).When two (or more) antisense compounds, or a combination of one or moreantisense compounds and one or more NABAs, are to be administeredsimultaneously in a treatment regime, one preferred composition is onecomprising a lipid vesicle, particularly a sterically stabilized lipidvesicle, comprising both (or all) of the compounds. In the context ofthe invention, the term "treatment regimen" is meant to encompasstherapeutic, palliative and prophylactic modalities.

1. Combinations of Antisense Compounds: Two or more antisense compoundscan be administered simultaneously as described above. Combinationtreatments can also be carried out by first (1) administering a firstcomposition comprising one or more antisense compounds targeted to oneor more CAMs (or a combination thereof with one or moreanti-inflammatory/immunosuppressive or chemotherapeutic agents) for afirst period of time and then (2) "switching" to administration of asecond composition comprising one or more antisense compounds targetedto one or more CAMs (or a combination thereof with one or moreanti-inflammatory or chemotherapeutic agents) for a second period oftime. Whether administered simultaneously or sequentially, preferredpairings of antisense compounds include those targeted to molecules thatmediate cellular adhesion, such as: (1) PECAM-1 and ICAM-1; (2) PECAM-1and VCAM-1; (3) PECAM-1 and ELAM-1; (4) PECAM-1 and a B7 protein, suchas B7-1 or B7-2; (5) ICAM-1 and VCAM-1; (6) ICAM-1 and ELAM-1; (7)ICAM-1 and a B7 protein; (8) VCAM-1 and ELAM-1; (9) VCAM-1 and a B7protein; (10) ELAM-1 and a B7 protein. Antisense compounds targeted toICAM-1, VCAM-1, ELAM-1 and B7 proteins are described in U.S. Pat. Nos.5,514,788 and 5,591,623, and copending U.S. patent application Ser. No.30 08/777,266, filed Dec. 31, 1996, all to Bennett et al. Becauseprotein kinase C (PKC) appears to be required for, or at least involvedin, PECAM-1 phosphorylation and activation (Zehnder et al., J. Biol.Chem., 1992, 267, 5243; Lastres et al., J. Immunol., 1994, 153, 4206;Shen et al., Am. J. Physiol., 1996, 270 (Heart Circ. Physiol., 39),H1624), combinations of the PECAM-1-targeted antisense compounds of theinvention and antisense compounds targeted to PKC may be particularlyeffective and are a preferred embodiment of the invention. Antisensecompounds targeted to PKC are described in U.S. Pat. Nos. 5,620,963 toCook et al. and 5,681,747 to Boggs et al. NABAs that inhibit PKC, suchas, e.g., staurosporin, may additionally or alternatively be used incombination with the antisense compounds of the invention. One skilledin the art can prepare and test more complex combinations of antisensecompounds, i.e., methods and compositions such as those described abovebut wherein three or more antisense compounds are administered.

2. Combinations with Chemotherapeutic Agents: For the purpose oftreating hyperproliferative disorders, the antisense compounds of theinvention can additionally or alternatively be used in combination withnon-antisense-based chemotherapeutic agents. Examples of such agentsthat can be used in combination with the antisense compounds of theinvention include but are not limited to daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, nitrogen mustards,methylcyclohexylnitrosurea, melphalan, cyclophosphamide,6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine,hydroxyurea, deoxycoformycin, 5-fluorouracil (5-FU),4-hydroxyperoxycyclophosphoramide, 5-fluorodeoxyuridine (5-FUdR),methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide,trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). (See,generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., pp.1206-1228, Berkow et al., eds., Rahay, N.J., 1987).

3. Combinations with Anti-Inflanmmatory/Immuno-suppressive Agents:Examples of non-antisense-based anti-inflammatory or immunosuppressiveagents that can be used in combination with the antisense compounds ofthe invention include but are not limited to salicylates; nonsteroidalanti-inflammatory drugs (NSAIDS), including indomethacin, ibuprofen,fenopofen, ketoprofen, naproxen, piroxicam, phenylbutazone,oxyphenbutazone, sulindac and meclofenamate; gold compounds, such asauranofin; D-penicillamine; cyclophosphamide; methotrexate;azathioprine; colchicine; hydroxychloroquine; corticotropin; steroidsand corticosteroids such as, for example, hydrocortisone,deoxyhydrocortisone, fludrocortisone, prednisolone, methylprednisolone,prednisone, triamcinolone, dexamethasone, betamethasone andparamethasone. See, generally, The Merck Manual of Diagnosis andTherapy, 15th Ed., pp. 1239-1267 and 2497-2506, Berkow et al., eds.,Rahay, N.J., 1987).

4. Combinations with Antibiotics: Some forms of arthritis result frominfection of an animal by a pathogen, such as a bacteria or spirochete,and subsequent immune system-mediated events, e.g., inflammation. Anobservation that may at least partially explain such undesirable immuneresponses is that extracts of the arthritis-related spirochete Borreliaburgdorferi induce the upregulation of several cellular adhesionmolecules (including ICAM-1, VCAM-1, E-selectin and P-selectin) onbEND.3 cells, an effect that is blocked by the anti-inflammatory agentprednisolone (Hurtenbach et al., Int. J. Immunopharmacol., 1996, 18,281; Boggemeyer et al., Cell. Adhes. Commun., 1994, 2, 145). The presentinvention provides a method of treating spirochetal infection of ananimal comprising administering one or more of the antisense compoundsof the invention to an animal, in a pharmaceutical preparation ifrequired.

If the undesired inflammation results from infection from a pathogen oneor more antibiotics may also be used in combination with an antisensecompound of the invention with or without an anti-inflammatory orimmunosuppressive agent. The choice of antibiotic(s) to be used willdepend on the nature of the pathogen(s) (i.e., bacteria such as cocci,bacilli, etc. or spirochetes). Such antibiotics include but are notlimited to nafcillin (gram-positive cocci) ; penicillin G (gram-negativecocci) ; gentamicin and/or piperacillin (gram-negative bacilli); and oneor more tetracyclines, one or more penicillin derivatives, erythromycin,doxycylcine, chloramphenicol and/or streptomycin (spirochetes such as,e.g., Borrelia burgdorferi and Treponema pallidum). See, generally, TheMerck Manual of Diagnosis and Therapy, 15th Ed., pp. 132-136, 236-243,1239-1267 and 1918-1919, Berkow et al., eds., Rahay, N.J., 1987). Oneskilled in the art will appreciate that, depending on the cause andstage of development of an undesired immune response resulting frompathogenic infection, it may be preferable in some instances toadminister, either simultaneously or sequentially, more complexcombinations. That is, the present invention encompasses more complexcompositions and treatment regimes such as those comprising, forexample, (1) one or more antisense compounds targeted to one or moreCAMs, (2) one or more antibiotics, and (3) one or moreanti-inflammatory/immunosuppressive NABAs.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  68    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2557 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Unknown    -     (ii) MOLECULE TYPE: mRNA    -     (iv) ANTI-SENSE: No    -      (x) PUBLICATION INFORMATION:              (A) AUTHORS: Newman,P.J.                   Berndt,M.C.                   Gorski,J.                   White,G.C.II - #.                   Lyman,S.                   Paddock,C.                   Muller,W.A.              (B) TITLE: PECAM-1 (CD3 - #1) cloning and relation to    #molecules of the immunoglobulin gene                   superfamily              (C) JOURNAL: Science              (D) VOLUME: 247              (E) ISSUE: 4947              (F) PAGES: 1219-1222              (G) DATE: 09-MAR-1990    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:    #              50GTGACC AGAGCAATTT CTGCTTTTCA CAGGGCGGGT    #             100TTGTGG GCAGTGCCTT CTGCTGAGCG AGTCATGGCC    #             150ACTGTG CCTGCAGTCT TCACTCTCAG GATGCAGCCG    #             200GGCCAC GATGTGGCTT GGAGTCCTGC TGACCCTTCT    #             250TTGAGG GTCAAGAAAA CTCTTTCACA ATCAACAGTG    #             300CTGCCG GACTGGACGG TGCAAAATGG GAAGAACCTG    #             350CGCGGA TGTCAGCACC ACCTCTCACG TCAAGCCTCA    #             400TCTATA AGGATGACGT GCTGTTTTAC AACATCTCCT    #             450GAGAGT TATTTTATTC CTGAAGTCCG GATCTATGAC    #             500ATGTAC TGTGATTGTG AACAACAAAG AGAAAACCAC    #             550TGTTGG TGGAAGGAGT GCCCAGTCCC AGGGTGACAC    #             600GCCATC CAAGGTGGGA TCGTGAGGGT CAACTGTTCT    #             650GGCCCC AATACACTTC ACAATTGAAA AACTTGAACT    #             700TCAAGC TGAAAAGAGA GAAGAATTCT CGAGACCAGA    #             750GAATTC CCCGTTGAGG AACAGGACCG CGTTTTATCC    #             800TAGGAT CATTTCTGGG ATCCATATGC AGACCTCAGA    #             850AACTGG TCACCGTGAC GGAATCCTTC TCTACACCCA    #             900CCCACC GGAATGATCA TGGAAGGAGC TCAGCTCCAC    #             950TCAAGT GACTCACCTG GCCCAGGAGT TTCCAGAAAT    #            1000ACAAGG CGATTGTGGC CCACAACAGA CATGGCAACA    #            1050GTCATG GCCATGGTGG AGCACAGTGG CAACTACACG    #            1100CAGCCG CATATCCAAG GTCAGCAGCA TCGTGGTCAA    #            1150TTTCCA AGCCCGAACT GGAATCTTCC TTCACACATC    #            1200AGACTG AACCTGTCCT GCTCCATCCC AGGAGCACCT    #            1250CATCCA GAAGGAAGAT ACGATTGTGT CACAGACTCA    #            1300TAGCCT CAAAGTCGGA CAGTGGGACG TATATCTGCA    #            1350AAAGTG GTCAAGAAAA GCAACACAGT CCAGATAGTC    #            1400CTCCCA GCCCAGGATT TCTTATGATG CCCAGTTTGA    #            1450AGACCA TCGAAGTCCG TTGCGAATCG ATCAGTGGAA    #            1500TACCAA CTTTTAAAAA CAAGTAAAGT TTTGGAGAAT    #            1550AAATGA TCCTGCGGTA TTCAAAGACA ACCCCACTGA    #            1600AGTGTG TTGCAGATAA TTGCCATTCC CATGCCAAAA    #            1650CTGAGG GTGAAGGTGA TAGCCCCGGT GGATGAGGTC    #            1700GTCAAG TAAGGTGGTG GAGTCTGGAG AGGACATTGT    #            1750TGAATG AAGGATCTGG TCCCATCACC TATAAGTTTT    #            1800GGCAAA CCCTTCTATC AAATGACCTC AAATGCCACC    #            1850CAAGCA GAAGGCTAGC AAGGAACAGG AGGGAGAGTA    #            1900TCAACA GAGCCAACCA CGCCTCCAGT GTCCCCAGAA    #            1950GTCAGA GTCATTCTTG CCCCATGGAA GAAAGGACTT    #            2000CATCGG AGTGATCATT GCTCTCTTGA TCATTGCGGC    #            2050TGAGGA AAGCCAAGGC CAAGCAGATG CCAGTGGAAA    #            2100GTACCA CTTCTGAACT CCAACAACGA GAAAATGTCA    #            2150AGCTAA CAGTCATTAC GGTCACAATG ACGATGTCAG    #            2200AACCAA TAAATGATAA TAAAGAGCCT CTGAACTCAG    #            2250GAAGTT CAAGTGTCCT CAGCTGAGTC TCACAAAGAT    #            2300CACAGA GACAGTGTAC AGTGAAGTCC GGAAAGCTGT    #            2350AAAGCA GATACTCTAG AACGGAAGGC TCCCTTGATG    #            2400AGGCCA GATGCACATC CCTGGAAGGA CATCCATGTT    #            2450TAATCC CTGTATTTCA AGACCTCTGT GCACTTATTT    #            2500CTCCCA CAGAACACAG CAATTCCTCA GGCTAAGCTG    #            2550CATCCT GCTAAGTTAA TGTTGGGTAG AAAGAGATAC    #        2557    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 2:    # 20               CCGG    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 3:    # 20               GAAA    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 4:    # 20               TCAG    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 5:    # 20               AAGA    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 6:    # 20               GCAG    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 7:    # 20               CCAC    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:    # 20               TGTC    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 9:    # 20               ATAA    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 10:    # 20               TGAG    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 11:    #20                TAAA    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 12:    #20                TTGC    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 13:    #20                CCGT    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 14:    #20                GGCC    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 15:    # 20               GCAC    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 16:    # 20               GTCT    - (2) INFORMATION FOR SEQ ID NO: 17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 17:    #                 22ATC AT    - (2) INFORMATION FOR SEQ ID NO: 18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 18:    #                 22TAT CT    - (2) INFORMATION FOR SEQ ID NO: 19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 19:    # 20               CATC    - (2) INFORMATION FOR SEQ ID NO: 20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 20:    # 20               TCTG    - (2) INFORMATION FOR SEQ ID NO: 21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 21:    # 20               TCAT    - (2) INFORMATION FOR SEQ ID NO: 22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 22:    # 20               GTTC    - (2) INFORMATION FOR SEQ ID NO: 23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 23:    # 20               CACT    - (2) INFORMATION FOR SEQ ID NO: 24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 24:    # 20               CCAC    - (2) INFORMATION FOR SEQ ID NO: 25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 25:    # 20               GCAG    - (2) INFORMATION FOR SEQ ID NO: 26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 26:    # 20               ATGG    - (2) INFORMATION FOR SEQ ID NO: 27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 27:    # 20               ATCG    - (2) INFORMATION FOR SEQ ID NO: 28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 28:    # 20               CATC    - (2) INFORMATION FOR SEQ ID NO: 29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 29:    # 20               ATGT    - (2) INFORMATION FOR SEQ ID NO: 30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2534 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Unknown    -     (ii) MOLECULE TYPE: mRNA    -     (iv) ANTI-SENSE: No    -      (x) PUBLICATION INFORMATION:              (A) AUTHORS: Xie,Y.M.                   Muller,W.A.              (B) TITLE: Molecular cl - #oning and adhesion    #of murine platelet/endothelial cell    #molecule 1 superfamily              (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.              (D) VOLUME: 90              (E) ISSUE: 12              (F) PAGES: 5569-5573              (G) DATE: 15-JUN-1993    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 30:    #              50GCCGGA AGACAGAACT AGCTGAGTGC TTCAGTCTGC    #             100ATTCAG CTGAGGTGGG CCTCAGTCGG CAGCAAAGAT    #             150GACTCA CGCTGGTGCT CTATGCAAGC CTCCAGGCTG    #             200ACCATC AACAGCATCC ATATGGAAAG CCTGCCATCA    #             250TGGGCA GCAACTGACC CTGGAGTGCC TTGTGGACAT    #             300AAAGCA GGTCTCAGCA CCGGGTGCTG TTCTATAAGG    #             350TATAAC GTCACCTCCA GGGAGCACAC CGAGAGCTAC    #             400TCGGGT CTTCCACTCC GGGAAGTACA AATGCACAGT    #             450AGGAAA AAACCACGAT TGAGTACGAG GTGAAGGTGC    #             500CCCAAG GTGACACTGG ACAAAAAGGA GGTGACAGAA    #             550GGTCAA TTGTTCCTTG CAAGAAGAAA AGCCACCGAT    #             600AAAAAT TAGAAGTGGG GACAAAGTTT GTCAAGCGAA    #             650TCCAAC GAGAACTTTG TGCTCATGGA ATTCCCCATT    #             700CGTGTT AGTGTTTCGC TGCCAAGCTG GGATCCTGTC    #             750AGGAGT CAGAACCCAT CAGGAGTGAA TACGTCACCG    #             800TCCACT CCCAAGTTTG AAATCAAGCC CCCTGGGATG    #             850CCAGCT GCACATTAGG TGCATAGTTC AAGTGACACA    #             900TTACAG AAATTATCAT CCAAAAAGAC AAGGCGATTG    #             950CAAAGC AGTGAAGCTG TCTACTCAGT CATGGCCATG    #            1000ACACTA CACCTGCAAA GTGGAATCAA ACCGTATCTC    #            1050TCATGG TCAACATAAC AGAGCTGTTT CCCAAGCCGA    #            1100TCCAGT CGTCTGGACC AAGGGGAGTT GTTGGACCTG    #            1150GGGCAC ACCTGTAGCC AACTTCACCA TCCAGAAGGA    #            1200CGCAGT ATCAGAATTT CAGCAAGATC GCCGAGGAGA    #            1250TACAGC TGTACTGCAG GCATCGGCAA AGTGGTCAAG    #            1300ACCGAT CCAGGTGTGC GAAATGCTCT CGAAGCCCAG    #            1350CCAAGT CTGAGATCAT AAAAGGACAT GCCATAGGCA    #            1400GAAAAT GGAACTGCAC CCATCACTTA CCACCTTATG    #            1450CTTCCA GACTCTCGAG GTGACCTCCA ATGACCCAGC    #            1500AGCCCA CCAGAGACAT GGAATACCAG TGCAGAGCGG    #            1550CACCCC GCGGTGTTCA GCGAGATCCT GAGGGTCAGG    #            1600GGATGA AGTTGTGATT TCCATCCTGT CGAGTAACGA    #            1650GTGAAA TGGTACTTCG GTGCTCTGTG AAAGAGGGGA    #            1700TTTCAG TTTTACAAAG AAAAGGAGGA CAGACCCTTC    #            1750GAATGA CACCCAAGCG TTTTGGCACA ACAAACAAGC    #            1800AAGGAC AGTACTACTG TACAGCCTCC AACAGAGCCA    #            1850AGTCCC CGAAGCAGCA CTCTTGCAGT CAGAGTCTTC    #            1900GAAAGG GCTCATTGCG GTGGTTGTCA TTGGAGTGGT    #            1950TAGTTG CAGCCAAATG CTACTTCCTG AGGAAAGCCA    #            2000CCCGTG GAGATGTCCA GGCCAGCTGC TCCACTTCTG    #            2050GAAGAT TTCTGAGCCT AGTGTGGAAG CCAACAGCCA    #            2100ATGTTT CTGGAAATGA TGCAGTAAAA CCCATAAATC    #            2150CAGAAC ATGGATGTAG AATACACAGA AGTGGAAGTG    #            2200TCACCA AGCTCTGGGA ACGAGAGCCA CAGAGACGGT    #            2250GGAAGG TCGACCCTAA TCTCATGGAA AACAGATACT    #            2300TCCCTT AATGGAACTT AAGACAGTCA AGGCAGTGCA    #            2350CGTCCA TGTCCCGAGA AGAGCAGCCG ATTCCTGGAT    #            2400GCACGT ATTTATGAGC CTGTCCCCAC CGAAAGCAGT    #            2450AGCCAT CCAATTTTAA ACCCCGCTGC CTTGTTCATG    #            2500ACTCAG AGGCGCTAGT TAATGGCCAG CCTGACCTAT    #      2534        GGGG TGAAAGGAGG TTCC    - (2) INFORMATION FOR SEQ ID NO: 31:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 31:    #                 22AGT AT    - (2) INFORMATION FOR SEQ ID NO: 32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 32:    #                 22AGC CT    - (2) INFORMATION FOR SEQ ID NO: 33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 33:    # 20               CAGG    - (2) INFORMATION FOR SEQ ID NO: 34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 34:    # 20               CAGC    - (2) INFORMATION FOR SEQ ID NO: 35:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 35:    # 20               TGAG    - (2) INFORMATION FOR SEQ ID NO: 36:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 36:    # 20               TCCA    - (2) INFORMATION FOR SEQ ID NO: 37:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 37:    # 20               TTCA    - (2) INFORMATION FOR SEQ ID NO: 38:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 38:    # 20               CATT    - (2) INFORMATION FOR SEQ ID NO: 39:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 39:    # 20               CTTC    - (2) INFORMATION FOR SEQ ID NO: 40:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 40:    # 20               CAAT    - (2) INFORMATION FOR SEQ ID NO: 41:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 41:    # 20               TCCA    - (2) INFORMATION FOR SEQ ID NO: 42:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 42:    # 20               TTCC    - (2) INFORMATION FOR SEQ ID NO: 43:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 43:    # 20               TCTT    - (2) INFORMATION FOR SEQ ID NO: 44:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 44:    # 20               TCGG    - (2) INFORMATION FOR SEQ ID NO: 45:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 45:    # 20               TGGG    - (2) INFORMATION FOR SEQ ID NO: 46:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 46:    # 20               CAGC    - (2) INFORMATION FOR SEQ ID NO: 47:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 47:    # 20               CAGG    - (2) INFORMATION FOR SEQ ID NO: 48:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 48:    # 20               CTCT    - (2) INFORMATION FOR SEQ ID NO: 49:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 49:    # 20               TTCT    - (2) INFORMATION FOR SEQ ID NO: 50:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 50:    # 20               TCCA    - (2) INFORMATION FOR SEQ ID NO: 51:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 51:    #                 22TGT GT    - (2) INFORMATION FOR SEQ ID NO: 52:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 52:    #                 22GGT GT    - (2) INFORMATION FOR SEQ ID NO: 53:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 53:    # 20               CTAC    - (2) INFORMATION FOR SEQ ID NO: 54:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 90 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 54:    #              50CAGATA CTCTAGAACG GAAGGCTCCC TTGATGGAAC    #    90            GATG CACATCCCTG GAAGGACATC    - (2) INFORMATION FOR SEQ ID NO: 55:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 80 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 55:    #              50CTCTAG AACGGAAGGC TCCCTTGATG GAACTTAGAC    #           80     CATC CCTGGAAGGA    - (2) INFORMATION FOR SEQ ID NO: 56:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 80 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 56:    #              50TGCATC TGGCCTTGCT GTCTAAGTTC CATCAAGGGA    #           80     TATC TGCTTTCCAC    - (2) INFORMATION FOR SEQ ID NO: 57:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 59 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 57:    #              50TGCATC TGGCCTTGCT GTCTAAGTTC CATCAAGGGA    #         59    - (2) INFORMATION FOR SEQ ID NO: 58:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 15 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 58:    #    15    - (2) INFORMATION FOR SEQ ID NO: 59:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 59:    #       12    - (2) INFORMATION FOR SEQ ID NO: 60:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 60:    #      13    - (2) INFORMATION FOR SEQ ID NO: 61:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 85 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 61:    #              50TATGAA CCTGCCCTGC TCCCACAGAA CACAGCAATT    #       85         CGGT TCTTAAATCC ATCCT    - (2) INFORMATION FOR SEQ ID NO: 62:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 59 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 62:    #              50CTGCTC CCACAGAACA CAGCAATTCC TCAGGCTAAG    #         59    - (2) INFORMATION FOR SEQ ID NO: 63:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 59 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: YES    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 63:    #              50CCTGAG GAATTGCTGT GTTCTGTGGG AGCAGGGCAG    #         59    - (2) INFORMATION FOR SEQ ID NO: 64:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 64:    #             27   TGAG GAATTGC    - (2) INFORMATION FOR SEQ ID NO: 65:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 65:    #      13    - (2) INFORMATION FOR SEQ ID NO: 66:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 66:    #                23TCAT AAA    - (2) INFORMATION FOR SEQ ID NO: 67:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: Yes    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 67:    #   17             T    - (2) INFORMATION FOR SEQ ID NO: 68:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 59 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (iv) ANTI-SENSE: No    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 68:    #              50TGATGG AACTTAGACA GCAAGGCCAG ATGCACATCC    #         59    __________________________________________________________________________

What is claimed is:
 1. An antisense compound up to 30 nucleobases inlength connected by covalent linkages, wherein said antisense compoundcomprises a nucleobase sequence specifically hybridizable with a regionof a nucleic acid encoding a human platelet endothelial cell adhesionmolecule 1, said nucleobase sequence being selected from a groupconsisting of SEQ ID NO: 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 19, 20,21, 22, 23, 24, 25 and 26, wherein said antisense compound interfereswith mRNA encoding human platelet endothelial cell adhesion molecule 1thereby decreasing expression of said human platelet endothelial celladhesion molecule
 1. 2. A pharmaceutical composition comprising one ormore of the antisense compounds of claim
 1. 3. An antisense compoundhaving a nucleobase sequence that specifically hybridizes to a sequencecontained within SEQ ID NO: 55 or
 68. 4. The antisense compound of claim3 wherein said nucleobase sequence is 5'-GTGCATC, 5'-TGGCC or SEQ IDNOS: 58, 59 or
 60. 5. An antisense compound having a nucleobase sequencethat specifically hybridizes to a sequence contained within SEQ ID NO:62, 64 or
 66. 6. The antisense compound of claim 5 wherein saidnucleobase sequence is SEQ ID NO: 65 or
 67. 7. An antisense compound upto 30 nucleobases in length connected by covalent linkages, wherein saidantisense compound comprises a nucleobase sequence specificallyhybridizable with a nucleic acid encoding a murine platelet endothelialcell adhesion molecule 1, said nucleobase sequence being selected from agroup consisting of SEQ ID NO: 33, 34, 36, 37, 38, 39, 40, 41, 42, 43 or44, wherein said antisense compound interferes with mRNA encoding murineplatelet endothelial cell adhesion molecule 1 thereby decreasingexpression of said murine platelet endothelial adhesion molecule 1.